Cd40 agonist antibody /type 1 interferon synergistic adjuvant combination, conjugates containing and use thereof as a therapeutic to enhance cellular immunity

ABSTRACT

A synergistic adjuvant is provided comprising synergistically effective amounts of at least one type 1 interferon and at least one CD40 agonist, wherein these moieties may be in the same or separate compositions. In addition, fusion proteins and DNA conjugates which contain a type 1 interferon/CD40 agonist/antigen combination are provided. The use of these compositions, protein and DNA conjugates as immune adjuvants for treatment of various chronic diseases such as HIV infection and for enhancing the efficacy of vaccines (prophylacetic and therapeutic) is also provided.

RELATED APPLICATIONS

This application relates to U.S. provisional application Ser. No.60/796,867 filed on May 3, 2006, 60/809,821 filed on Jun. 1, 2006 and60/842,009 filed on Sep. 5, 2006, all of which applications areincorporated by reference in their entirety. Also, the applicationrelates to U.S. provisional application 60/777,569 filed on Mar. 1, 2006which application is also incorporated by reference herein.

FIELD OF THE INVENTION

The invention generally relates to synergistic adjuvant combinationswhich may be used to enhance immunity in subjects in need thereof. Moreparticularly, the invention relates to a specific synergistic adjuvantcombination comprising (i) a type 1 interferon and (ii) a CD40 agonist,e.g., an agonistic anti-CD40 antibody or a CD40L polypeptide or CD40Lfragment or CD40L containing conjugate, and optionally further including(iii) a target antigen.

Additionally, the invention relates to novel protein or DNA conjugatescomprising or encoding said synergistic adjuvant combination such asprotein and DNA conjugates which comprise or encode (i) a CD40 agonisticantibody or a soluble CD40L protein or CD40L fragment or CD40L conjugateand (ii) a type 1 interferon and optionally (iii) a desired antigen.

Still further the invention provides novel immune therapies comprisingthe administration of such synergistic adjuvant combinations or DNA orprotein conjugates to enhance antigen specific cellular immunity, e.g.,CD8+ immunity. Specifically, the use of compositions comprising thesenovel adjuvant combinations and/or or polypeptide conjugates and DNAconjugates for treating various chronic diseases including cancer, forexample CD40 antigen expressing tumors, and for treating infectiousdiseases such as HIV infection, autoimmune diseases, allergic andinflammatory diseases, and for potentiating the efficacy of vaccines isalso taught.

Also the invention provides novel methods for alleviating the toxicityof CD40 agonists such as CD40L polypeptides and conjugates or agonisticCD40 antibodies by co-administering such CD40 agonist with an amount ofa type 1 interferon sufficient to alleviate or prevent toxicity, e.g.,liver toxicity that would otherwise result upon administration of theCD40 agonist alone. This facilitates the administration of CD40 agonistsat therapeutic dosages which would otherwise be precluded based ontoxicity.

BACKGROUND OF THE INVENTION

The body's defense system against microbes as well as the body's defenseagainst other chronic diseases such as those affecting cellproliferation is mediated by early reactions of the innate immune systemand by later responses of the adaptive immune system. Innate immunityinvolves mechanisms that recognize structures which are for examplecharacteristic of the microbial pathogens and that are not present onmammalian cells. Examples of such structures include bacterialliposaccharides, (LPS) viral double stranded DNA, and unmethylated CpGDNA nucleotides. The effector cells of the innate immune response systemcomprise neutrophils, macrophages, and natural killer cells (NK cells).In addition to innate immunity, vertebrates, including mammals, haveevolved immunological defense systems that are stimulated by exposure toinfectious agents and that increase in magnitude and effectiveness witheach successive exposure to a particular antigen. Due to its capacity toadapt to a specific infection or antigenic insult, this immune defensemechanism has been described as adaptive immunity. There are two typesof adaptive immune responses, called humoral immunity, involvingantibodies produced by B lymphocytes, and cell-mediated immunity,mediated by T lymphocytes.

Two types of major T lymphocytes have been described, CD8+ cytotoxiclymphocytes (CTLs) and CD4 helper cells (Th cells). CD8+ T cells areeffector cells that, via the T cell receptor (TCR), recognize foreignantigens presented by class I MHC molecules on, for instance, virally orbacterially infected cells. Upon recognition of foreign antigens, CD8+cells undergo an activation, maturation and proliferation process. Thisdifferentiation process results in CTL clones which have the capacity ofdestroying the target cells displaying foreign antigens. T helper cellson the other hand are involved in both humoral and cell-mediated formsof effector immune responses. With respect to the humoral, or antibodyimmune response, antibodies are produced by B lymphocytes throughinteractions with Th cells. Specifically, extracellular antigens, suchas circulating microbes, are taken up by specialized antigen-presentingcells (APCs), processed, and presented in association with class IImajor histocompatibility complex (MHC) molecules to CD4+ Th cells. TheseTh cells in turn activate B lymphocytes, resulting in antibodyproduction. The cell-mediated, or cellular, immune response, incontrast, functions to neutralize microbes which inhabit intracellularlocations, such as after successful infection of a target cell. Foreignantigens, such as for example, microbial antigens, are synthesizedwithin infected cells and resented on the surfaces of such cells inassociation with Class I MHC molecules. Presentation of such epitopesleads to the above-described stimulation of CD8+ CTLs, a process whichin turn also stimulated by CD4+ Th cells. Th cells are composed of atleast two distinct subpopulations, termed Th1 and Th2 cells. The Th1 andTh2 subtypes represent polarized populations of Th cells whichdifferentiate from common precursors after exposure to antigen.

Each T helper cell subtype secretes cytokines that promote distinctimmunological effects that are opposed to one another and thatcross-regulate each other's expansion and function. Th1 cells secretehigh amounts of cytokines such as interferon (IFN) gamma, tumor necrosisfactor-alpha (TNF-alpha), interleukin-2 (IL-2), and IL-12, and lowamounts of IL-4. Th1 associated cytokines promote CD8+ cytotoxic Tlymphocyte T lymphocyte (CTL) activity and are most frequentlyassociated with cell-mediated immune responses against intracellularpathogens. In contrast, Th2 cells secrete high amounts of cytokines suchas IL-4, IL-13, and IL-10, but low IFN-gamma, and promote antibodyresponses. Th2 responses are particularly relevant for humoralresponses, such as protection from anthrax and for the elimination ofhelminthic infections.

Whether a resulting immune response is Th1 or Th2-driven largely dependson the pathogen involved and on factors in the cellular environment,such as cytokines. Failure to activate a T helper response, or thecorrect T helper subset, can result not only in the inability to mount asufficient response to combat a particular pathogen, but also in thegeneration of poor immunity against reinfection. Many infectious agentsare intracellular pathogens in which cell-mediated responses, asexemplified by Th1 immunity, would be expected to play an important rolein protection and/or therapy. Moreover, for many of these infections ithas been shown that the induction of inappropriate Th2 responsesnegatively affects disease outcome. Examples include M tuberculosis, S.mansoni, and also counterproductive Th2-like dominated immune responses.Lepromatous leprosy also appears to feature a prevalent, butinappropriate, Th2-like response. HIV infection represents anotherexample. There, it has been suggested that a drop in the ratio ofTh1-like cells to other Th cell populations can play a critical role inthe progression toward disease symptoms.

As a protective measure against infectious agents, vaccination protocolsfor protection from some microbes have been developed. Vaccinationprotocols against infectious pathogens are often hampered by poorvaccine immunogenicity, an inappropriate type of response (antibodyversus cell-mediated immunity), a lack of ability to elicit long-termimmunological memory, and/or failure to generate immunity againstdifferent serotypes of a given pathogen. Current vaccination strategiestarget the elicitation of antibodies specific for a given serotype andfor many common pathogens, for example, viral serotypes or pathogens.Efforts must be made on a recurring basis to monitor which serotypes areprevalent around the world. An example of this is the annual monitoringof emerging influenza A serotypes that are anticipated to be the majorinfectious strains.

To support vaccination protocols, adjuvants that would support thegeneration of immune responses against specific infectious diseasesfurther have been developed. For example, aluminum salts have been usedas a relatively safe and effective vaccine adjuvants to enhance antibodyresponses to certain pathogens. One of the disadvantages of suchadjuvants is that they are relatively ineffective at stimulating acell-mediated immune response and produce an immune response that islargely Th2 biased.

It is now widely recognized that the generation of protective immunitydepends not only on exposure to antigen, but also the context in whichthe antigen is encountered. Numerous examples exist in whichintroduction of a novel antigen into a host in a non-inflammatorycontext generates immunological tolerance rather than long-term immunitywhereas exposure to antigen in the presence of an inflammatory agent(adjuvant) induces immunity. (Mondino et al., Proc. Natl. Acad. Sci.,USA 93:2245 (1996); Pulendran et al., J. Exp. Med. 188:2075 (1998);Jenkins et al., Immunity 1:443 (1994); and Kearney et al., Immunity1:327 (1994)).

A naturally occurring molecule well known to regulate adaptive immunityis CD40. CD40 is a member of the TNF receptor superfamily and isessential for a spectrum of cell-mediated immune responses and requiredfor the development of T cell dependent humoral immunity (Aruffo et al.,Cell 72:291 (1993); Farrington et al., Proc Natl Acad. Sci., USA 91:1099(1994); Renshaw et al., J Exp Med 180:1889 (1994)). In its natural role,CD40-ligand expressed on CD4+ T cells interacts with CD40 expressed onDCs or B cells, promoting increased activation of the APC and,concomitantly, further activation of the T cell (Liu et al. SeminImmunol 9:235 (1994); Bishop et al., Cytokine Growth Factor Rev 14:297(2003)). For DCs, CD40 ligation classically leads to a response similarto stimulation through TLRs such as activation marker upregulation andinflammatory cytokine production (Quezada et al. Annu Rev Immunol 22:307(2004); O'Sullivan B and Thomas R Crit Rev Immunol 22:83 (2003)) Itsimportance in CD8 responses was demonstrated by studies showing thatstimulation of APCs through CD40 rescued CD4-dependent CD8+ T cellresponses in the absence of CD4 cells (Lefrancois et al., J Immunol.164:725 (2000); Bennett et al., Nature 393:478 (1998); Ridge et al.,Nature 393:474 (1998); Schoenberger et al., Nature 393:474 (1998); .This finding sparked much speculation that CD40 agonists alone couldpotentially rescue failing CD8+ T cell responses in some diseasesettings.

Other studies, however, have demonstrated that CD40 stimulation aloneinsufficiently promotes long-term immunity. In some model systems,anti-CD40 treatment alone insufficiently promoted long-term immunity.Particularly, anti-CD40 treatment alone can result in ineffectiveinflammatory cytokine production, the deletion of antigen-specific Tcells (Mauri et al. Nat Med 6:673 (2001); KedI et al. Proc Natl AcadSci., USA 98:10811 (2001)) and termination of B cell responses (Ericksonet al., J Clin Invest 109:613 (2002)). Also, soluble trimerized CD40ligand has been used in the clinic as an agonist for the CD40 pathwayand what little has been reported is consistent with the conclusion thatstimulation of CD40 alone fails to reconstitute all necessary signalsfor long term CD8+ T cell immunity (Vonderheide et al., J Clin Oncol19:3280 (2001)).

Various agonistic antibodies have been reported by different groups. Forexample, one mAb CD40.4 (5c3) (PharMingen, San Diego Calif.) has beenreported to increase the activation between CD40 and CD40L byapproximately 30-40%. (Schlossman et al., Leukocyte Typing, 1995,1:547-556). Also, Seattle Genetics in U.S. Pat. No. 6,843,989 allege toprovide methods of treating cancer in humans using an agonisticanti-human CD40 antibody. Their antibody is purported to deliver astimulatory signal, which enhances the interaction of CD40 and CD40L byat least 45% and enhances CD40L-mediated stimulation and to possess invivo neoplastic activity. They derive this antibody from S2C6, anagonistic anti-human CD40 antibody previously shown to deliver stronggrowth-promoting signals to B lymphocytes. (Paulie et al., 1989, J.Immunol. 142:590-595).

Because of the role of CD40 in innate and adaptive immune responses,CD40 agonists including various CD40 agonistic antibodies have beenexplored for usage as vaccine adjuvants and in therapies whereinenhanced cellular immunity is desired. Recently, it was demonstrated bythe inventor and others that immunization with antigen in combinationwith some TLR agonists and anti-CD40 treatment (combined TLR/CD40agonist immunization) induces potent CD8+ T cell expansion, elicting aresponse 10-20 fold higher than immunization with either agonist alone(Ahonen et al., J Exp Med 199:775 (2004)). This was the firstdemonstration that potent CD8+ T cell responses can be generated in theabsence of infection with a viral or microbial agent. Antigen specificCD8+ T cells elicited by combined TLR/CD40 agonist immunizationdemonstrate lytic function, gamma interferon production, and enhancedsecondary responses to antigenic challenge. Synergistic activity withanti-CD40 resulting in the induction of CD8+ T cell expansion has beenshown with agonists of TLR1/6, 2/6, 3, 4, 5, 7 and 9.

To increase the effectiveness of an adaptive immune response, such as ina vaccination protocol or during a microbial infection, it is thereforeimportant to develop novel, more effective, vaccine adjuvants. Thepresent invention satisfies this need and provides other advantages aswell.

Also, it is important to develop effective immune adjuvants which areeffective at doses which do not elicit adverse side effects such asliver toxicity. Particularly it has been reported by Vanderheide et al.,J Clin. Oncol. 25(7)876-8833(March 2007) that a 0.3 mg/kg is the maximumtolerated dose for an exemplified agonistic antibody and that higherdoses may elicit side effects including venous thromboembolism, grade 3headache, cytokine release resulting in toxic effects such as chills andthe like, and transient liver toxicity. Also, it has been reported byVanderheide et al., J Clin. Oncol. 19(23):4351-3 (2001) that the maximumtolerated dose for a hCD40L polypeptide described therein was 0.1mg/kg/day and that when the polypeptide was administered at higher dosesof 0.15 mg/kg/day they observed liver toxicity characterized by grade 3or 4 liver transaminase elevated levels in subjects treated.

SUMMARY OF THE INVENTION

This invention in one embodiment involves the discovery that certainmoieties in combination upregulate CD70 on dendritic cells and elicit asynergistic effect on immunity, e.g., they promote Th1 cellular immunityand CD8 T cell immune responses. Particularly, the invention involvesthe discovery that type 1 interferons and CD40 agonists, such asagonistic CD40 antibodies or CD40L polypeptides or CD40L conjugates,when administered in combination in the same or separate compositions,and further optionally in combination with a desired antigen, elicit asynergistic effect on immunity by inducing CD70 expression on CD8+dendritic cells and moreover elicit potent expansion of CD8+ T cells andenhanced Th1 immunity.

Based on this discovery, the present invention provides novel adjuvantcombinations that can be administered to subjects in need thereof as ameans of enhancing immunity. Also, this adjuvant combination can beadded to vaccines or administered in conjunction therewith in order toenhance the efficacy thereof.

Related to the said discovery, the invention also provides nucleic acidconstructs that encode (i) a type 1 interferon and (ii) a CD40 agonistthat optionally may further include (iii) a nucleic acid sequenceencoding a desired antigen, which nucleic acid constructs, whenadministered to a host in need thereof, optionally in conjunction withan antigen, elicit a synergistic effect on immunity. Such CD40 agonistsinclude by way of example CD40 agonistic antibodies and CD40 agonisticantibody fragments, as well as soluble CD40L and CD40L fragments andconjugates and derivatives thereof such as oligomeric CD40Lpolypeptides, e.g., trimeric CD40L polypeptides and conjugatescontaining.

Also, the present invention provides polypeptide conjugates comprising(i) at least one type 1 interferon, (ii) at least one CD40 agonist, e.g.a CD40 agonistic antibody or CD40L polypeptide or CD40L fragment orconjugate or derivative thereof such as an oligomeric CD40L or conjugatecontaining, and optionally (iii) an antigen, wherein these moieties maybe directly or indirectly linked, in any order, and elicit a synergisticeffect on immunity on administration to a subject in need thereof.

More specifically, this invention provides nucleic acid constructscontaining (i) a gene or genes encoding an agonistic anti-human CD40antibody, or human CD40L polypeptide or fragment, conjugate orderivative thereof, and (ii) a gene encoding a human type 1 interferon,e.g. human alpha or human beta interferon and optionally (iii) a geneencoding an antigen against which an enhanced cellular immune responseis desirably elicited.

Also more specifically the invention provides novel polypeptideconstructs comprising (i) at least one agonistic anti-human CD40antibody or a human CD40L polypeptide or fragment thereof that agonizeshuman CD40/CD40L, a human alpha or beta interferon, and optionally atleast one antigen against which an enhanced cellular immune response isdesirably elicited.

Still further, the invention provides adjuvant polypeptide compositionscomprising synergistically effective amount of (i) a type 1 interferon,preferably alpha or beta interferon, (ii) a CD40 agonist, preferably anagonistic CD40 antibody or a monomeric or oligomeric soluble CD40 Lpolypeptide or fragment or conjugate thereof, and optionally (iii) oneor more antigens.

Also, the invention relates to the discovery that the toxicity of CD40agonists can potentially be alleviated if the CD40 agonist isadministered in conjunction with a type 1 interferon or a TLR agonist.Thereby, the invention provides for more effective CD40 agonisttherapies as the CD40 agonist can be administered at higher dosages thanheretofore described. For example the MTD (maximum tolerated dosage) ofCD40L polypeptide if co-administered with a type 1 interferon or a TLRagonist may exceed 0.1 mg/kg/day by at least 1.5 fold, more preferablyby at least 2-5 fold, or even 10-fold or more thereby permitting theCD40L polypeptide to be administered at MTD amounts ranging from atleast about 0.15 mg/kg/day to 1.0 mg/kg/day or higher. This will resultin more effective CD40L therapies such as in the treatment of CD40associated malignancies and other treatments disclosed herein. Inaddition the present invention will reduce toxicity of CD40 agonistantibody therapies and facilitate the administration of CD40 agonistantibody dosages higher than heretofore suggested. Particularly, asnoted above it has been reported that the MTD for an agonistic CD40Lantibody reported by Vonderheide et al., J Clin. Immunol. 25(7):876-883(2007) was 0.3 mg/kg and that dosages in excess resulted in transientliver toxicity, venous thromboembolism, grade 3 headaches and cytokinerelease and associated toxicity and adverse side effects such a feverand chills. Co-administration of the CD40 agonist antibody inassociation with type 1 interferon or a TLR agonist potentially allowsfor the MTD antibody amount to be substantially increased, e.g. by1.5-15 or even 5-10 fold without adverse effects. Thereby the MTD amountfor the CD40 agonistic antibody may be increased to about 0.45 mg/kg toabout 3.0 mg/kg or even higher. Thus the invention includes theco-administration of a CD40 agonist with an amount of type 1 interferonor TLR agonist sufficient to reduce toxic effects such as liver toxicitythat would otherwise potentially result at the particular CD40 agonistdosage amount.

In addition the invention provides novel therapies comprisingadministration of any of the foregoing protein or DNA conjugates orsynergistic adjuvant protein containing compositions. These therapiesinclude the use thereof as immune agonists (adjuvants) such as tosynergistically enhance the efficacy of vaccines and for treatingconditions wherein enhanced immunity is desired such as cancer,infectious conditions, autoimmune conditions, allergy, inflammatoryconditions and gene therapy.

As noted above and shown infra it has been surprisingly discovered thatthe afore-described novel adjuvant combination or protein or DNAconjugates encoding elicits a synergistic effect on immunity relative tothe administration of the CD40 agonist or the type 1 interferon aloneand/or potentially reduces or prevents adverse side effects such asliver toxicity. Such reduced toxicity can e.g., be determined based onthe effect of the immunostimulatory combination on liver transaminaselevels. This synergism is apparently obtained because the adjuvantcombinations of the invention surprisingly induce (upregulate) CD70expression on CD8+ dendritic cells in vivo, and thereby induce thepotent expansion of CD8+ T cells in vivo.

At least based on these surprising synergistic effects on dendriticcells, and on CD8+ T cell immunity and Th1 immunity, compositionscontaining these adjuvant combinations, nucleic acid constructs, orpolypeptide conjugates may be administered to a host in need of thereofas a means of:

(i) generating enhanced (exponentially better) primary and memory CD8+ Tcell responses relative to immunization with either agonist alone;

(ii) inducing the exponential expansion of antigen-specific CD8+ Tcells, and/or

(iii) generating protective immunity.

Accordingly, these adjuvants combinations which may comprise proteincompositions, or nucleic acid constructs encoding or polypeptideconjugates containing may be used in treating any disease or conditionwherein the above-identified enhanced cellular immune responses aretherapeutically desirable, especially infectious diseases, proliferativedisorders such as cancer, allergy, autoimmune disorders, inflammatorydisorders, and other chronic diseases wherein enhanced cellular immunityis a desired therapeutic outcome. Preferred applications of theinvention include especially the treatment of infectious disorders suchas HIV infection and cancer.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 shows CD8+ T cell expansion following combined TLR/CD40 agonistimmunized is variably dependent on IFN α/β. WT (top row) and IFNαβRKO(bottom row) were immunized with ovalbumin peptide, anti-CD40 and theindicated TLR agonists. 7 days late, the ovalbumin specific T cellresponses were measured in the spleen by tetramer staining and FACSanalysis. Numbers in the upper right quadrant indicate the percentage oftetramer staining cells out of the total CD8+ T cells.

FIG. 2 shows CD4 depletion of IFNαβRKO hosts restores the CD8+ T cellresponse after immunization with the IFNαβ-dependent TLR agonist incombination with anti-CD40. WT and IFNαβRKO mice, CD40-depleted ornon-depleted as indicated were immunized with HSV-1 peptide, anti-CD40,and polylC as described above. 7 days later, the HSV-1 specific responsewas determined by tetramer (A) and polylC IFNgamma (B) staining PBLs).

FIG. 3 shows anti-IFN blocks polylC/CD40 mediated CD8 response which isrecovered by CD4− depletion. Mice were immunized against ovalbumin(combined polylC/alphaCD40) with and without anti-IFN and/or CD4depletion. Day 7 PBLs were analyzed by tetramer staining as describedabove, for the antigen-specific T cells.

FIG. 4 shows the CD8+ T cell response in CD4-depleted, IFNαβRKO hostsfollowing combined TLR/CD40 immunization is largely dependent on CD70.IFNαβRKO mice were depleted of CD4 cells and immunized with HSV-1peptide, polylC and anti-CD40 as described above. Mice were injectedwith anti-TNF ligand antibodies as in FIG. 6. Day 7 PBLs were analyzedby tetramer staining.

FIG. 5 shows IFN and CD40 synergize to elicit exponential CD8+ T cellexpansion. Mice were challenged as described above. 7 days after initialantigen challenge, PBLs were analyzed by tetramer staining.

FIG. 6 contains the results of an experiment relating to combinedadministration of a type 1 interferon and an agonistic antibody showingthat this combination induces CD70 expression on CD8+ dendritic cells invivo whereas administration of either alone does not. Mice were injectedwith anti-CD40 antibody alone, polylC as a positive control, recombinanttype 1 interferon (1×10⁷ U) or anti-CD40+IFN. 18 hours later spleen DCswere isolated and analyzed for their expression of CD70. The numbers inthe upper right quadrant indicate the mean fluorescence intensity ofCD70 staining. The data reveal that, similar to CD40/polylC injection,CD40/IFN similarly increase the expression of CD70 on CD8+ DCs.

FIG. 7 contains an experiment showing the effect of combined type 1interferon administration and an agonistic CD40 antibody on CD70expression on CD8+ DCs in vivo. The results show that only theimmunostimulatory combinations and not CD40 agonist or IFN alone induceCD70 expression on DCs.

FIG. 8 contains an experiment that analyzed the percentage ofantigen-specific (ovalbumin T cells) in mice administered ant-CD40,IFNalpha, polylC/CD40, IFNalpha and anti-CD70 or IFNalpha/CD40 atvarious decreasing IFN doses.

FIG. 9 similarly to the experiment in FIG. 7 shows combined TLR/CD40agonist challenge induces CD70 expression only on DCs expressing thetargeted TLR in IFNαβRKO mice. IFNαβRKO mice were injected withanti-CD40 alone (aCD40) or in combination with poylC (+polylC) orPam3Cys (+Pam3Cys). Pam3Cys is a TLR2 agonist and PolylC is a TLR3agonist. 24 hours later the spleen DCs were isolated and stained forCD70 expression as described above. CD8+ DCs express TLR2 and 3 whileCD11b+DCs express TLR2 but not TLR3. The data suggest that in theabsence of IFNαβ signaling only DCs stimulated directly through both TLRand CD40 are able to increase CD70 expression.

FIG. 10 contains an experiment comparing the effect of IL-2/CD40 agonistcombination and IFNalpha/CD40 agonist combination on the percentage ofantigen-specific (ovalbumin) T cells from PBLs. The results containedtherein show that the IL-2/CD40 agonist combination does not elicit acomparable synergistic effect on CD8+ T cell immunity as theIFNalpha/CD40 agonist combination.

FIG. 11 contains an experiment in C57BI/6 mice with injected melanomacells showing that the IFNalpha/CD40 agonist combination increasedsurvival time in this metastatic melanoma animal model.

FIG. 12 contains an experiment showing the subject combination adjuvanttherapy with CD40 agonist and IFN alpha in a C57BI/6 animal model formetastatic lung cancer protects the mice from metastatic lung cancer asshown by a reduced number of metastatic nodules in animals treated withthe adjuvant combination.

FIG. 13 contains an experiment wherein TIL analysis was effected inC57BI/6 mice inoculated with B16.F10 melanoma cells treated with thesubject adjuvant combination and appropriate controls. The miceadministered the subject adjuvant combination revealed increased numbersof TILs as shown by the data in the Figure.

FIG. 14 contains an experiment that shows that the subject CD40agonist/IFN combination therapy generates antigen-specific effector Tcells that infiltrate the lungs of tumor bearing mice (C57BI/6 miceinoculated with B16.F10 melanoma cells)

FIGS. 15A and 15B contain light and heavy chain sequences for theexemplary CD40 agonistic antibody (FGK.45) used in the examples.

FIG. 16 contains a schematic showing construction of a DNA construct forexpression of a CD40 agonistic antibody-antigen-type 1 IFN conjugate ina baculovirus expression system according to the invention. Thisconstruct will result in the expression of an anti-CD40 antibody linkedto an antigen of choice (e.g. HIV gag) and to a type 1 interferon (alphainterferon).

FIG. 17 contains a construct for producing CD40 ab-antigen-type 1 IFNconjugate according to the invention in a baculovirus expression systemand a construct for producing a vector for use in DNA immunization.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the invention generally relates to synergistic adjuvantcombinations and use thereof. Prior to discussing the invention in moredetail, the following definitions are provided. Otherwise all termsshould be construed as they would be a person of skill in the art.

In the present invention, the term “agonist” includes any entity thatdirectly binds and activates a receptor or which indirectly activates areceptor by forming a complex with another entity that binds thereceptor or by causing the modification of another compound thatthereupon directly binds and activates the receptor.

The term “CD40 agonist” in particular includes any entity which agonizesCD40/CD40L and/or which increases one or more CD40 or CD40L associatedactivities. This includes by way of example CD40 agonistic antibodies,fragments thereof, soluble CD40L and fragments and derivatives thereofsuch as oligomeric (e.g., bivalent, trimeric CD40L), and fusion proteinscontaining and variants thereof produced by recombinant or proteinsynthesis. In addition such CD40 agonists include small molecules, andCD40 aptamers which comprise RNA or DNA molecules that can besubstituted for antibodies. Techniques for the production and usethereof as antigen binding moieties may be found e.g., in U.S. Pat. Nos.5,475,046; 5,720,163; 5,589,332; and 5,741,679. These patents areincorporated by reference in their entirety herein.

in the present invention the term “CD40L” or “CD154” as it alternativelyknown in the art includes all mammalian CD40L's, e.g., human, rat,non-human primate, murine as well as fragments, variants, oligomers, andconjugates thereof that bind to at least the corresponding mammalianCD40 polypeptide, e.g., human CD40. In the present invention theadministered CD40L may comprise a CD40L polypeptide or a DNA encodingsaid CD40L polypeptide. Such CD40L polypeptides and DNAs include inparticular native CD40L sequences and fragments, variants, and oligomersthereof as disclosed in Immunex U.S. Pat. No. 6,410,711; U.S. Pat. No.6,391,637; U.S. Pat. No. 5,981,724; U.S. Pat. No. 5,961,974 and USpublished application No. 20040006006 all of which patents andapplication and the CD40L sequences disclosed therein are incorporatedby reference in their entirety herein.

In the present invention the term 4-1 BB agonist includes any entitythat agonizes the 4-1 BB receptor such as agonistic 4-1 BB antibodiesand 4-1 mM polypeptides and conjugates thereof. Such agonistspotentially can be co-administered with a type 1 interferon or TLRagonist to elicit a synergistic effects on immunity.

In the present invention the term “type 1 interferon” encompasses anytype 1 interferon which elicits an enhanced CD8+ immune response whenadministered proximate to or in combination with a CD40 agonist. Thisincludes alpha interferons, beta interferons and other types ofinterferons classified as type 1 interferons. Particularly, thisincludes epsilon interferon, zeta interferon, and tau interferons suchas tau 1 2, 3, 4, 5, 6, 7, 8, 9, and 10; Also, this includes variantsthereof such as fragments, consensus interferons which mimic thestructure of different type 1 interferon molecules such as alphainterferons, PEGylated versions thereof, type 1 interferons with alteredglycosylation because of recombinant expression or mutagenesis, and thelike. Those skilled in the art are well aware of different type 1interferons including those that are commercially available and in useas therapeutics. Preferably the type 1 interferon will comprise a humantype 1 interferon and most preferably a human alpha interferon.

The term “synergistic adjuvant” or “synergistic combination” in thecontext of this invention includes the combination of two immunemodulators such as a receptor agonist, cytokine, adjuvant polypeptide,that in combination elicit a synergistic effect on immunity relative toeither administered alone. Particularly, this application disclosessynergistic combinations that comprise at least one type 1 interferonand a CD40 agonist or a TLR agonist and a CD40 agonist or a TLR agonistor type 1 interferon and a 4-1BB agonist. These synergistic combinationsupon administration together or proximate to one another elicit agreater effect on immunity, e.g., relative to when the CD40 agonist ortype 1 interferon is administered in the absence of the other moiety.For example, the greater effect may be evidenced by the upregulation ofCD70 on dendritic cells in vivo that does not occur when either immunemodulator or agonist is administered alone.

“Co-administration” in the present invention refers to theadministration of different entities such as a type 1 interferon and aCD40 agonist or a protein conjugate or DNA conjugate or conjugatesencoding for same under conditions such that the entities, e.g., CD40agonist and the type 1 interferon elicit a synergistic effect onimmunity and e.g., result in the upregulation of CD70 on dendritic cellsand/or reduce adverse side effects such as liver toxicity. The moietiesmay be administered in the same or different compositions which ifseparate are administered proximate to one another, generally within 24hours of each other and more typically within about 1-8 hours of oneanother, and even more typically within 1-4 hours of each other or closeto simultaneous administration. The relative amounts are dosages thatachieve the desired synergism. In addition the agonists if administeredin the form of DNA conjugates may be comprised on the same or differentvector, such as a plasmid or recombinant viral vector such as anadenoviral or vaccinia vector.

“Vaccine” refers to a composition which on administration alone or inconjunction with the adjuvant combination of the invention results in anantigen-specific effect on immunity. This includes prophylaceticvaccines which confer protection and therapeutic vaccines.

the term “antibody” refers to an intact antibody or a binding fragmentthereof that competes with the intact antibody for specific binding.Binding fragments are produced by recombinant DNA techniques, or byenzymatic or chemical cleavage of intact antibodies. Binding fragmentsinclude Fab, Fab′, F(ab)2, Fv and single chain antibodies. This includesin particular chimeric, human, humanized, bispecific, and non-humanantibodies. Additionally, such antibodies and fragments include variantsthereof which are altered to affect one or more properties such ascleavage, glycosylation, effector function, and the like.

As noted above, there is a significant need for the development andimplementation of new vaccine adjuvants and/or adjuvant formulationsthat are able to generate potent antigen-specific T cell immunity andwhich are not subject to undesired side effects such as liver toxicity.

The present invention satisfies this need by providing novel adjuvantsthat may be administered alone or in conjunction with existing vaccinesin order to enhance their efficacy. These adjuvants will typicallyinclude at least one type 1 interferon, preferably alpha or beta humaninterferon, at least one CD40 agonist (anti-CD40 antibody or fragmentthereof) or a soluble CD40L polypeptide.

The present invention provides methods of eliciting enhanced cellularimmune responses in subjects in need thereof by administering thecombination of at least one CD40 agonist, preferably a CD40 agonisticantibody or soluble CD40L, a type 1 interferon, such as human alpha orbeta interferon and optionally a target antigen, e.g., a tumor antigen,autoantigen, allergen or a viral antigen. These moieties elicit asynergistic effect on cellular immunity by eliciting CD70 expression onCD8+ dendritic cells. Specifically, this combination induces thefollowing: (i) exponential increase in generation of primary and memoryCD8+ T cell response than either agonist alone (ii) exponentialexpansion of CD8+ T cells and (iii) should elicit protective immunity.As shown infra the induction of CD70 expression on CD8+ dendritic cellsdoes not occur when either the CD40 agonistic antibody or the type 1interferon are administered alone. Therefore, the CD40 agonist/IFNcombination surprisingly synergizes inducing CD70 expression on CD8+ DCsand potent expansion of CD8+ T cells in vivo.

Related to this discovery the present invention further provides DNAconstructs encoding a novel synergistic agonistic polypeptide conjugatethat promotes cellular immunity comprising (i) a DNA encoding a CD40agonist preferably a CD40 agonistic antibody or fragment thereof or asoluble CD40L or fragment or derivative and (ii) a DNA encoding a type 1interferon, e.g., alpha or beta interferon and which constructpreferably further includes (iii) a DNA encoding a desired antigen.

The present invention further provides synergistic protein conjugatesthat elicit a synergistic effect on cellular immunity comprising a CD40agonist, preferably a agonistic CD40 antibody or fragment or a fragmentof CD40L, a type 1 interferon, and optionally a desired target antigen.

The invention further provides compositions containing these DNAconstructs which when administered to a host, preferably a human, may beused to generate enhanced antigen specific cellular immune responses.

The present invention further provides expression vectors and host cellscontaining a DNA construct encoding said novel synergistic agonisticpolypeptide combination comprising (i) a DNA or DNAs encoding a specificCD40 agonist, preferably a agonistic CD40 antibody or antibody fragmentor a fragment of CD40L, (ii) a DNA or DNAs encoding a type 1 interferon,preferably alpha or beta interferon and (iii) preferably a DNA thatencodes an antigen against which enhanced antigen specific cellularimmune response are desirably elicited, e.g. a viral or tumor antigen.

Also, the invention provides methods of using said vectors and hostcells to produce a composition containing said novel synergisticIFN/CD40 agonist/antigen polypeptide conjugate, preferably an agonisticCD40 ab/antigen/type 1 interferon polypeptide conjugate.

Further the invention provides methods of administering said DNAconstructs or compositions and vehicles containing to a host in which anantigen specific cellular immune response is desirably elicited, forexample a person with a chronic disease such as cancer or an infectiousor allergic disorder under conditions which preferably reduce oreliminate undesired side effects such as liver toxicity.

Still further the invention provides compositions comprising said novelsynergistic IFN/CD40 agonist antigen polypeptide conjugates which aresuitable for administration to a host in order to elicit an enhancedantigen-specific cellular immune response.

Also, the present invention provides compositions suitable fortherapeutic use comprising the combination of at least one type 1interferon, at least one CD40 agonist, and optionally a target antigenwhich elicit a synergistic effect on cellular immunity when administeredto a host in need of such administration.

Also, the invention provides novel methods of immunotherapy comprisingthe administration of said novel synergistic agonist-antigen polypeptideconjugate or a DNA encoding said polypeptide conjugate or a compositionor compositions containing at least one type 1 interferon, at least oneCD40 agonist and optionally at least one target antigen to a host inneed of such treatment in order to elicit an enhanced (antigen specific)cellular immune response. In preferred embodiments these compositionsand conjugates will be administered to a subject with or at risk ofdeveloping a cancer, an infection, particularly a chronic infectiousdisease e.g., involving a virus, bacteria or parasite; or an autoimmune,inflammatory or allergic condition. For example the invention may beused to elicit antigen specific cellular immune responses against HIV.HIV is a well recognized example of a disease wherein protectiveimmunity almost certainly will require the generation of potent andlong-lived cellular immune responses against the virus.

Also, the invention provides methods of enhancing the efficacy ofvaccines, particularly vaccines intended to induce a protective cellularimmune response by combining or co-administering the subject synergisticadjuvant combination which upregulates CD70 on dendritic cells. In thepreferred embodiment such adjuvant will comprise the specific adjuvantsdisclosed herein and optionally may further comprise another adjuvantsuch as a TLR, e.g., a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10 or TLR11. Ideally, this additional adjuvant will furtherinduce CD70 expression by dendritic cells and result in further enhancedimmune responses in a subject in need thereof.

The present invention is an extension of the inventors' priordemonstration that the immunization with antigen in the presence ofagonists for both a toll-like receptor (TLR) and CD40 (combined TLR/CD40agonist immunization) elicits a vigorous expansion of antigen specificCD8+ T cells. The response elicited from this form of vaccination isexponentially greater than the response elicited by either agonistalone, and is far superior to vaccination by conventional methods.Combined TLR/CD40 agonist immunization has been observed to producepotent primary and secondary CD8+ T cell responses, achieving 50-70%antigen specific T cells in the circulation after only 2 immunizations.However, unlike the inventors' prior invention, the present synergisticcombination comprises the combination of a type 1 interferon and a CD40agonist or a 4-1 BB agonist. It has been surprisingly found that bothTLR/CD40 agonistic antibody combinations and type 1 interferon/CD40agonistic antibody combinations induce CD70 expression on CD8+ DCs andthereby elicit potent expansion of CD8+ T cells in vivo. Thus, the CD40pathway is seemingly integrated with both the TLR and the type 1 IFNsignaling pathways providing for the induction of synergisticallyenhanced DC activation and thereby potent induction of antigen specificcellular immunity.

To elicit a synergistic effect on cellular immunity, the CD40 agonist,the type 1 interferon and an antigen if present are preferablyadministered as discrete polypeptide moieties which may be jointly orseparately administered, in either order, substantially proximate orsimultaneous to one another under conditions that result in the desiredsynergistic effect on immunity. Whether synergism is obtained may bedetected by various means, e.g., based on the upregulation of CD70expression on dendritic cells under the administration conditions.Alternatively, these moieties may be administered as a singlepolypeptide fusion or conjugates containing these two or three discreteentities or administered in the form of a DNA conjugate or conjugatesencoding said two or three discrete entities. The latter two embodimentsof the invention are advantageous in the context of a polypeptide or DNAbased vaccine since potentially only one active agent will need to beformulated and administered to a subject in need of treatment, forexample an individual with HIV infection or cancer.

The present invention satisfies this need by providing novel adjuvantsthat may be administered alone or in conjunction with existing vaccinesin order to enhance their efficacy. These adjuvants will typicallyinclude at least one type 1 interferon, preferably alpha or beta humaninterferon, at least one CD40 agonist (anti-CD40 antibody or fragmentthereof or soluble CD40L polypeptide) and preferably at least oneantigen against which enhanced antigen-specific cellular immunity isdesirably elicited such as a tumor antigen or viral antigen. In apreferred embodiment of the invention these polypeptide moieties will becontained in a single polypeptide conjugate or will be encoded by anucleic acid construct which upon expression in vitro in a host cell orin vivo upon administration to a host results in the expression of saidagonist and antigen polypeptides or the expression of a conjugatecontaining these polypeptides.

The administered amounts of the type 1 interferon and the CD40 agonist,e.g., an agonistic CD40 antibody will comprise amounts that incombination or co-administration yield a synergistic effect by inducingCD70 expression on dendritic cells and enhanced numbers of antigenspecific CD8+ T cells. Ideally, the dosage will not result in adverseside effects such as liver toxicity which can be detected e.g., based onliver transaminase levels. With respect to the type 1 interferon, theamount may vary from about 1×10³ units of activity (U) to about 1×10¹⁰U, more typically from about 10⁴ U to about 10⁸ U. The amount of theagonistic antibody or CD40L polypeptide may vary from about 0.00001grams to about 5 grams, more typically from about 0.001 grams to about 1gram. As noted above, a preferred MTD will exceed 0.3 mg/kg and mayrange from about 0.45 mg/kg to about 3 mg/kg. If the therapeutic methodinvolves the administration of an antigen this may be administered atamounts ranging from about 0.0001 grams to about 50 grams, moretypically from about 0.1 grams to about 10 grams. As noted, thesemoieties may be administered in the same or different formulations. Ifadministered separately the moieties may be administered in any order,typically within several hours of each other, more typicallysubstantially proximate in time.

As noted, the CD40 agonist includes any moiety that agonizes theCD40/CD40L interaction. Typically these moieties will be CD40 agonisticantibodies or agonistic CD40L polypeptides. As discussed, theseantibodies include by way of example human antibodies, chimericantibodies, humanized antibodies, bispecific antibodies, scFvs, andantibody fragments that specifically agonize the CD40/CD40L bindinginteraction. Most preferably the antibody will comprise a chimeric,fully human or humanized CD40 antibody.

Human CD40L and other mammalian CD40L polypeptides are widely known andavailable including soluble forms thereof, oligomeric CD40L polypeptidessuch as trimeric CD40L originally reported by Immunex (now Amgen). Also,the sequence of human and murine CD40L is known and is commerciallyavailable. (See Immunex patents incorporated by reference supra). Asnoted above the CD40L dose will typically be at least 0.1 mg/kg/day andmore typically from at least about 0.15 to 1.0 mg/kg/day. The MTD willbe selected such that adverse side effects such as liver toxicity andincreased liver transaminase levels are not observed or are minimized ornegligible relative to when the CD40L polypeptide is administered in theabsence of the type 1 interferon or a TLR agonist.

As noted, the type 1 interferon can be any type 1 interferon or variantor fragment that elicits a synergistic effect on cellular immunity whenadministered proximate to or in combination with a CD40 agonist. Suchinterferons may include alpha interferon, beta interferon, interferontaus such as tau 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, interferon omega,interferon epsilon, interferon zeta and the like, especially variantsand fragments thereof. This especially includes PEGylated interferonsand consensus interferons and interferons with altered (non-native oraglycosylated) glycosylation.

While it has been previously reported by the inventors and others thatTLR agonists synergize with anti-CD40 agonists resulting in a profoundenhancement of CD8+ T cell immunity; these prior studies would not havesuggested that a type 1 interferon and a CD40 agonist such as anagonistic antibody would also yield synergistic effects on cellularimmunity. Surprisingly, the inventors have discovered that the CD40pathway is integrated with both the TLR and type 1 IFN signalingpathways for the induction of DC activation potent cellular immunity.Further, these earlier studies did not reveal the role of CD70 in thisprocess.

Also, the prior studies would not have suggested the subject DNA orpolypeptide conjugates since the prior studies involving TLRagonist/CD40 agonist combinations have required the separateadministration of the antigen, the TLR agonist and the CD40 agonist. Bycontrast this invention in some embodiments provides DNA constructs andbipartite or tripartite polypeptides that comprise two or threedifferent moieties or a DNA encoding these two or three moieties in asingle DNA or polypeptide molecule, e.g., a conjugate containing a CD40agonistic antibody, alpha interferon and an antigen. This shouldsimplify the use thereof for prophylacetic or therapeutic vaccinepurposes and or for enhancing cellular immunity in the treatment ofdiseases wherein enhanced cellular immunity is desired such as cancer orautoimmune condition (since only one molecular entity will need to beformulated in pharmaceutically acceptable form and administered). Thisis particularly advantageous in the context of treatment of a chronicdiseases or conditions wherein large amounts of adjuvant may be requiredfor effective prophylacetic or therapeutic immunity.

Combined IFN/CD40 agonist immunization, using only molecular reagents,uniquely generates CD8+ T cell responses of a magnitude that werepreviously only obtainable after challenge with an infectious agent(Ahonen et al., J Exp Med 199:775 (2004)). Thus, this invention providesfor the development of potent vaccines against HIV and other chronicinfectious diseases involving viruses, bacteria, fungi or parasites aswell as proliferative diseases such as cancer, autoimmune diseases,allergic disorders, and inflammatory diseases where effective treatmentrequires the quantity and quality of cellular immunity that onlycombined IFN (type 1)/CD40 agonist immunization or other adjuvantcombinations that upregulate CD70 expression on dendritic cells iscapable of generating.

APPLICATIONS OF THE INVENTION

The invention exemplifies herein both protein and DNA based vaccinescomprising the combination of (i) at least one CD40 agonist, e.g., anagonistic anti-CD40 ab or CD40L polypeptide, (ii) optionally at leastone target antigen (e.g., HIV Gag) and (iii) at least one Type 1Interferon (e.g. alpha interferon). HIVGag40 is an appropriate modelantigen because HIV is a chronic infectious disease wherein an enhancedcellular immune response has significant therapeutic potential. However,the invention embraces the construction of conjugates as describedcontaining any antigen against which an enhanced cellular immuneresponse is therapeutically desirable. In a preferred embodiment atleast one target antigen is comprised in the administered compositioncontaining at least one type 1 interferon, and at least one CD40 agonistor is contained in a polypeptide conjugate containing these moieties oris encoded by a DNA conjugate encoding these moieties. However, in someembodiments a conjugate containing type 1 interferon and the anti-CD40antibody may be administered separate from the antigen, or the host maybe naturally exposed to the antigen. Additionally, in some embodimentsall three moieties, i.e., the anti-CD40 antibody, the type 1 interferonand the antigen may be co-administered as separate discrete entities.Preferably all these moieties are administered substantiallyconcurrently in order to achieve the desired synergistic enhancement incellular immunity without adverse side effects such as liver toxicity,venous thromboembolism, cytokine toxicity, and/or headache. However,these moieties may be administered in any order that elicits asynergistic effect on cellular immunity resulting in enhanced CD8+ Tcell expansion and induction of CD70 expression on CD8+ DCs.

Exemplary antigens include but are not limited to bacterial, viral,parasitic, allergens, autoantigens and tumor associated antigens. If aDNA based vaccine is used the antigen will typically be encoded by asequence the administered DNA construct. Alternatively, if the antigenis administered as a conjugate the antigen will typically be a proteincomprised in the administered conjugate. Still further, if the antigenis administered separately from the CD40 agonist and the type 1interferon moieties the antigen can take any form. Particularly, theantigen can include protein antigens, peptides, whole inactivatedorganisms, and the like.

Specific examples of antigens that can be used in the invention includeantigens from hepatits A, B, C or D, influenza virus, Listeria,Clostridium botulinum, tuberculosis, tularemia, Variola major(smallpox), viral hemorrhagic fevers, Yersinia pestis (plague), HIV,herpes, pappilloma virus, and other antigens associated with infectiousagents. Other antigens include antigens associated with a tumor cell,antigens associated with autoimmune conditions, allergy and asthma.Administration of such an antigen in conjunction with the subjectagonist combination type 1 interferon and an anti-CD40 antibody can beused in a therapeutic or prophylacetic vaccine for conferring immunityagainst such disease conditions.

In some embodiments the methods and compositions can be used to treat anindividual at risk of having an infection or has an infection byincluding an antigen from the infectious agent. An infection refers to adisease or condition attributable to the presence in the host of aforeign organism or an agent which reproduce within the host. A subjectat risk of having an infection is a subject that is predisposed todevelop an infection. Such an individual can include for example asubject with a known or suspected exposure to an infectious organism oragent. A subject at risk of having an infection can also include asubject with a condition associated with impaired ability to mount animmune response to an infectious agent or organism, for example asubject with a congenital or acquired immunodeficiency, a subjectundergoing radiation or chemotherapy, a subject with a burn injury, asubject with a traumatic injury, a subject undergoing surgery, or otherinvasive medical or dental procedure, or similarly immunocompromisedindividual.

Infections which may be treated or prevented with the vaccinecompositions of this invention include bacterial, viral, fungal, andparasitic. Other less common types of infection also include arerickettsiae, mycoplasms, and agents causing scrapie, bovine spongiformencephalopathy (BSE), and prion diseases (for example kuru andCreutzfeldt-Jacob disease). Examples of bacteria, viruses, fungi, andparasites that infect humans are well know. An infection may be acute,subacute, chronic or latent and it may be localized or systemic.Furthermore, the infection can be predominantly intracellular orextracellular during at least one phase of the infectious organism'sagent's life cycle in the host.

Bacteria infections against which the subject vaccines and methods maybe used include both Gram negative and Gram positive bacteria. Examplesof Gram positive bacteria include but are not limited to Pasteurellaspecies, Staphylococci species, and Streptococci species. Examples ofGram negative bacteria include but are not limited to Escherichia coli,Pseudomonas species, and Salmonella species. Specific examples ofinfectious bacteria include but are not limited to Heliobacter pyloris,Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria spp. (forexample M. tuberculosis, M. avium, M. intracellilare, M. kansaii, M.gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseriameningitidis, Listeria monocytogeners, Streptococcus pyogenes, (group AStreptococcus), Streptococcus agalactiae(Group B Streptococcus),Streptococcus (viridans group), Streptococcus faecalis, streptococcusbovis, Streptococcus (aenorobic spp.), Streptococcus pneumoniae,pathogenic Campylobacter spp., Enterococcus spp., Haemophilusinfluenzae, Bacillus anthracis, Corynebacterium diptheriae,Corynebacterium spp., Erysipelothrix rhusiopathie, Clostridiumperfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasteurella multocida, Bacteroides spp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelii.

Examples of viruses that cause infections in humans include but are notlimited to Retroviridae (for example human deficiency viruses, such asHIV-1 (also referred to as HTLV-III), HIV-II, LAC or IDLV-III/LAV orHIV-III and other isolates such as HIV-LP, Picornaviridae (for examplepoliovirus, hepatitis A, enteroviruses, human Coxsackie viruses,rhinoviruses, echoviruses), Calciviridae (for example strains that causegastroenteritis), Togaviridae (for example equine encephalitis viruses,rubella viruses), Flaviviridae (for example dengue viruses, encephalitisviruses, yellow fever viruses) Coronaviridae (for examplecoronaviruses), Rhabdoviridae (for example vesicular stomata viruses,rabies viruses), Filoviridae (for example Ebola viruses) Paramyxoviridae(for example parainfluenza viruses, mumps viruses, measles virus,respiratory syncytial virus), Orthomyxoviridae (for example influenzaviruses), Bungaviridae (for example Hataan viruses, bunga viruses,phleoboviruses, and Nairo viruses), Arena viridae (hemorrhagic feverviruses), Reoviridae (for example reoviruses, orbiviruses, rotaviruses),Bimaviridae, Hepadnaviridae (hepatitis B virus), Parvoviridae(parvoviruses), Papovaviridae (papilloma viruses, polyoma viruses),Adenoviridae (adenoviruses), Herpeviridae (for example herpes simplexvirus (HSV) I and II, varicella zoster virus, pox viruses) andIridoviridae (for example African swine fever virus) and unclassifiedviruses (for example the etiologic agents of Spongiformencephalopathies, the agent of delta hepatitis, the agents of non-A,non-B hepatitis (class 1 enterally transmitted; class 2 parenterallytransmitted such as Hepatitis C); Norwalk and related viruses andastroviruses).

Examples of fungi include Aspergillus spp., Coccidoides immitis,Cryptococcus neoformans, Candida albicans and other Candida spp.,Blastomyces dermatidis, Histoplasma capsulatum, Chlamydia trachomatis,Nocardia spp., and Pneumocytis carinii.

Parasites include but are not limited to blood-borne and/or tissueparasites such as Babesia microti, Babesi divergans, Entomoebahistolytica, Giarda lamblia, Leishmania tropica, Leishmania spp.,Leishmania braziliensis, Leishmania donovdni, Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Toxoplasmagondii, Trypanosoma gambiense and Trypanosoma rhodesiense (Africansleeping sickness), Trypanosoma cruzi (Chagus' disease) and Toxoplasmagondii, flat worms, and round worms.

As noted this invention embraces the use of the subject synergisticcombination or protein or DNA conjugates containing or encoding thissynergistic combination in treating proliferative diseases such ascancers. Cancer is a condition of uncontrolled growth of cells whichinterferes with the normal functioning of bodily organs and systems. Asubject that has a cancer is a subject having objectively measurablecancer cells present in the subjects' body. A subject at risk ofdeveloping cancer is a subject predisposed to develop a cancer, forexample based on family history, genetic predisposition, subject exposedto radiation or other cancer-causing agent. Cancers which migrate fromtheir original location and seed vital organs can eventually lead to thedeath of the subject through the functional deterioration of theaffected organ. Hematopoietic cancers, such as leukemia, are able toout-compete the normal hematopoietic compartments in a subject therebyleading to hematopoietic failure (in the form of anemia,thrombocytopenia and neutropenia), ultimately causing death.

A metastasis is a region of cancer cells, distinct from the primarytumor location, resulting from the dissemination of cancer cells fromthe primary tumor to other parts of the body. At the time of diagnosisof the primary tumor mass the subject may be monitored for the presenceof metastases. Metastases are often detected through the sole orcombined use of magnetic resonance imaging (MRI), computed tomography(CT), scans, blood and platelet counts, liver function studies, chest—X-rays and bone scans in addition to the monitoring of specificsymptoms.

The compositions, protein conjugates and DNA vaccines of the inventioncan be used to treat a variety of cancers or subjects at risk ofdeveloping cancer, including CD40 expressing and non-expressing cancersby the inclusion of a tumor-associated-antigen (TAA), or DNA encoding.This is an antigen expressed in a tumor cell. Examples of such cancersinclude breast, prostate, lung, ovarian, cervical, skin, melanoma,colon, stomach, liver, esophageal, kidney, throat, thyroid, pancreatic,testicular, brain, bone and blood cancers such as leukemia, chroniclymphocytic leukemia, and the like. The vaccination methods of theinvention can be used to stimulate an immune response to treat a tumorby inhibiting or slowing the growth of the tumor or decreasing the sizeof the tumor. A tumor associated antigen can also be an antigenexpressed predominantly by tumor cells but not exclusively.

Additional cancers include but are not limited to basal cell carcinoma,biliary tract cancer, bladder cancer, bone cancer, brain and centralnervous system (CNS) cancer, cervical cancer, choriocarcinoma,colorectal cancers, connective tissue cancer, cancer of the digestivesystem, endometrial cancer, esophageal cancer, eye cancer, head and neckcancer, gastric cancer, intraepithelial neoplasm, kidney cancer, larynxcancer, liver cancer, lung cancer (small cell, large cell), lymphomaincluding Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma;neuroblastoma; oral cavity cancer (for example lip, tongue, mouth andpharynx); ovarian cancer; pancreatic cancer; retinoblastoma;rhabdomyosarcoma; rectal cancer; cancer of the respiratory system;sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer;uterine cancer; cancer of the urinary system; as well as othercarcinomas and sarcomas.

The compositions, protein conjugates, and DNA s of the invention canalso be used to treat autoimmune diseases such as multiple sclerosis,rheumatoid arthritis, type 1 diabetes, psoriasis or other autoimmunedisorders. Other autoimmune disease which potentially may be treatedwith the vaccines and immune adjuvants of the invention include Crohn'sdisease and other inflammatory bowel diseases such as ulcerativecolitis, systemic lupus eythematosus (SLE), autoimmuneencephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis,Goodpasture's syndrome, pemphigus, Graves disease, autoimmune hemolyticanemia, autoimmune thrombocytopenic purpura, scleroderma withanti-collagen antibodies, mixed connective tissue disease, polypyositis,pernicious anemia, idiopathic Addison's disease, autoimmune associatedinfertility, glomerulonephritis) for example crescenticglomerulonephritis, proliferative glomerulonephritis), bullouspemphigoid, Sjogren's syndrome, psoriatic arthritis, insulin resistance,autoimmune diabetes mellitus (type 1 diabetes mellitus; insulindependent diabetes mellitus), autoimmune hepatitis, autoimmunehemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmunehepatitis, autoimmune hemophilia, autoimmune lymphoproliferativesyndrome, autoimmune uveoretinitis, and Guillain-Bare syndrome.Recently, arteriosclerosis and Alzheimer's disease have been recognizedas autoimmune diseases. Thus, in this embodiment of the invention theantigen will be a self-antigen against which the host elicits anunwanted immune response that contributes to tissue destruction and thedamage of normal tissues.

The compositions, protein conjugates and DNA vaccines of the inventioncan also be used to treat asthma and allergic and inflammatory diseases.Asthma is a disorder of the respiratory system characterized byinflammation and narrowing of the airways and increased reactivity ofthe airways to inhaled agents. Asthma is frequently although notexclusively associated with atopic or allergic symptoms. Allergy isacquired hypersensitivity to a substance (allergen). Allergic conditionsinclude eczema, allergic rhinitis, or coryza, hay fever, bronchialasthma, urticaria, and food allergies and other atopic conditions. Anallergen is a substance that can induce an allergic or asthmaticresponse in a susceptible subject. There are numerous allergensincluding pollens, insect venoms, animal dander, dust, fungal spores,and drugs.

Examples of natural and plant allergens include proteins specific to thefollowing genera: Canine, Dermatophagoides, Felis, Ambrosia, Lotium,Cryptomeria, Alternaria, Alder, Alinus, Betula, Quercus, Olea,Artemisia, Plantago, Parietaria, Blatella, Apis, Cupressus, Juniperus,Thuya, Chamaecyparis, Periplanet, Agopyron, Secale, Triticum, Dactylis,Festuca, Poa, Avena, Holcus, Anthoxanthum, Arrhenatherum, Agrostis,Phleum, Phalaris, Paspalum, Sorghum, and Bromis.

It is understood that the compositions, protein conjugates and DNAvaccines of the invention can be combined with other therapies fortreating the specific condition, e.g., infectious disease, cancer orautoimmune condition. For example in the case of cancer the inventivemethods may be combined with chemotherapy or radiotherapy.

Methods of making compositions as vaccines are well known to thoseskilled in the art. The effective amounts of the protein conjugate orDNA can be determined empirically, but can be based on immunologicallyeffective amounts in animal models. Factors to be considered include theantigenicity, the formulation, the route of administration, the numberof immunizing doses to be administered, the physical condition, weight,and age of the individual, and the like. Such factors are well known tothose skilled in the art and can be determined by those skilled in theart (see for example Paoletti and McInnes, eds., Vaccines, from Conceptto Clinic: A Guide to the Development and Clinical Testing of Vaccinesfor Human Use CRC Press (1999). As disclosed herein it is understoodthat the subject DNAs or protein conjugates can be administered alone orin conjunction with other adjuvants. Additionally, the subject adjuvantscan be added to or administered in conjunction with existing vaccines inorder to potentiate their efficacy. For example, these adjuvants may beused to potentiate the efficacy of viral vaccines such as the recentlyapproved HPV vaccine for cervical cancer. Also, they may be combinedwith other adjuvants.

The DNAs and protein conjugates of the invention can be administeredlocally or systemically by any method known in the art including but notlimited to intramuscular, intravenous, intradermal, subcutaneous,intraperitoneal, intranasal, oral or other mucosal routes. Additionalroutes include intracranial (for example intracisternal, orintraventricular), intraorbital, ophthalmic, intracapsular, intraspinal,and topical administration. The adjuvants and vaccine compositions ofthe invention can be administered in a suitable, nontoxic pharmaceuticalcarrier, or can be formulated in microcapsules or a sustained releaseimplant. The immunogenic compositions of the invention can beadministered multiple times, if desired, in order o sustain the desiredcellular immune response. The appropriate route, formulation, andimmunization schedule can be determined by one skilled in the art.

In the methods of the invention, in some instances the antigen and aType 1 IFN/CD40 agonist conjugate may be administered separately orcombined in the same formulation. In some instances it may be useful toinclude several antigens. These compositions may be administeredseparately or in combination in any order that achieve the desiredsynergistic enhancement of cellular immunity. Typically, thesecompositions are administered within a short time of one another, i.e.within about several days or hours of one another, most typically withinabout a half hour to an hour to facilitate the treatment regimen.

In some instances, it may be beneficial to include a moiety in theconjugate or the DNA which facilitates affinity purification. Suchmoieties include relatively small molecules that do not interfere withthe function of the polypeptides in the conjugate. Alternatively, thetags may be removable by cleavage. Examples of such tags includepoly-histidine tags, hemagglutinin tags, maltase binding protein,lectins, glutathione-S transferase, avidin and the like. Other suitableaffinity tags include FLAG, green fluorescent protein (GFP), myc, andthe like.

The subject adjuvant combinations and protein or DNA conjugates will beadministered with a physiologically acceptable carrier such asphysiological saline. The composition may also include another carrieror excipient such as buffers, such as citrate, phosphate, acetate, andbicarbonate, amino acids, urea, alcohols, ascorbic acid, phospholipids,proteins such as serum albumin, ethylenediamine tetraacetic acid, sodiumchloride or other salts, liposomes, mannitol, sorbitol, glycerol and thelike. The agents of the invention can be formulated in various ways,according to the corresponding route of administration. For example,liquid formulations can be made for ingestion or injection, gels orprocedures can be made for ingestion, inhalation, or topicalapplication. Methods for making such formulations are well known and canbe found in for example, “Remington's Pharmaceutical Sciences,” 18^(th)Ed., Mack Publishing Company, Easton Pa.

As noted the invention embraces DNA based vaccines. These DNAs may beadministered as naked DNAs, or may be comprised in an expression vector.Furthermore, the subject nucleic acid sequences may be introduce into acell of a graft prior to transplantation of the graft. This DNApreferably will be humanized to facilitate expression in a humansubject.

The subject polypeptide conjugates may further include a “marker” or“reporter”. Examples of marker or reporter molecules include betalactamase, chloramphenicol acetyltransferase, adenosine deaminase,aminoglycoside phosphotransferase, dihydrofolate reductase, hygromycinB-phosphotransferase, thymidine kinase, lacZ, and xanthine guaninephosphoribosyltransferase et al.

The subject nucleic acid constructs can be contained in any vectorcapable of directing its expression, for example a cell transduced withthe vector. The inventors exemplify herein a baculovirus vector as theyhave much experience using this vector. Other vectors which may be usedinclude T7 based vectors for use in bacteria, yeast expression vectors,mammalian expression vectors, viral expression vectors, and the like.Viral vectors include retroviral, adenoviral, adeno-associated vectors,herpes virus, simian virus 40, and bovine papilloma virus vectors.

Prokaryotic and eukaryotic cells that can be used to facilitateexpression of the subject polypeptide conjugates include by way ofexample microbia, plant and animal cells, e.g., prokaryotes such asEscherichia coli, Bacillus subtilis, and the like, insect cells such asSf21 cells, yeast cells such as Saccharomyces, Candida, Kluyveromyces,Schizzosaccharomyces, and Pichia, and mammalian cells such as COS,HEK293, CHO, BHK, NIH 3T3, HeLa, and the like. One skilled in the artcan readily select appropriate components for a particular expressionsystem, including expression vector, promoters, selectable markers, andthe like suitable for a desired cell or organism. The selection and useof various expression systems can be found for example in Ausubel etal., “Current Protocols in Molecular Biology, John Wiley and Sons, NewYork, N.Y. (1993); and Pouwels et al., Cloning Vectors: A LaboratoryManual”:, 1985 Suppl. 1987). Also provided are eukaryotic cells thatcontain and express the subject DNA constructs.

In the case of cell transplants, the cells can be administered either byan implantation procedure or with a catheter-mediated injectionprocedure through the blood vessel wall. In some cases, the cells may beadministered by release into the vasculature, from which the cellssubsequently are distributed by the blood stream and/or migrate into thesurrounding tissue.

The subject polypeptide conjugates or the DNA constructs contain orencode an agonistic anti-CD40 antibody or CD40L or fragment thereof thatspecifically binds or agonizes the binding of CD40 and CD40L, preferablymurine or human CD40. As used herein, the term “antibody” is used in itsbroadest sense to include polyclonal and monoclonal antibodies, as wellas antigen binding fragments thereof. This includes for example Fab,F(ab′)2, Fd and Fv fragments.

In addition the term “antibody” includes naturally antibodies as well asnon-naturally occurring antibodies such as single chain antibodies,chimeric antibodies, bifunctional and humanized antibodies. Preferredfor use in the invention are chimeric, humanized and fully humanantibodies. Methods for synthesis of chimeric, humanized, CDR-grafted,single chain and bifunctional antibodies are well known to those skilledin the art. In addition, agonistic antibodies specific to CD40 arewidely known and available and can be made by immunization of a suitablehost with a CD40 antigen, preferably human CD40.

The use of an anti-mouse CD40 antibody (FGK45) is exemplified in theexamples. This antibody was selected because anti-human CD40 antibodiesdo not specifically bind murine CD40 and the in vivo studies were inrodents. In the case of human therapy the selected agonistic CD40antibody will specifically bind human CD40. Agonistic CD40 antibodiesspecific to human CD40 are also known in the art and may be produced byknown methods. Alternatively, the CD40 agonist may comprise a fragmentof CD40L or a fusion protein containing that agonizes the interaction ofhuman CD40 and CD40L.

As noted the synergistic combinations of the invention contain at leastone type 1 interferon or a fragment or variant thereof that synergizeswith a CD40 agonist to induce CD70 expression on CD8+ DCs and elicitpotent expansion of CD8+ T cells in vivo. This includes by way ofexample alpha interferon, beta interferon, omega interferon, taointerferon, zeta interferon and epsilon interferon, et al as well asfunctional variants and fragments thereof.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.

Inventors' Rationale

As discussed above, all TLR agonists tested to date synergize withanti-CD40 for the induction of CD8⁺ T cell immunity. However, it wasobserved that some TLR agonist/anti-CD40 combinations (for TLRs 3, 7, 9)display a profound dependence upon type I interferon (IFNαβ) forenhancing CD8⁺ T cell expansion whereas other TLR/CD40 agonistcombinations (for TLRs 2 and 5) do not. Surprisingly, the depletion ofCD4 cells eliminates the IFNαβ requirement for generating CD8⁺ T cellresponses from TLR3-or-7/CD40-agonist combinations. Collectively thesedata suggested to the inventors a role for both IFNαβ and CD4 cells inregulating the CD8⁺ T cell response following combined TLR/CD40-agonistimmunization.

Based on these observations, the inventors hypothesized that theinduction of TNF ligand(s) on DCs is either dependent or independent ofIFNαβ, and that this determines the subsequent dependency of the CD8+ Tcell response on IFNαβ. Because the IFNαβ-dependent CD8⁺ T cell responsecan be recovered by CD4 depletion, it was hypothesized that either theexpression of CD70 on DCs, or the CD8+ T cell response, is negativelyinfluenced by regulatory T cells. We thereby proposed a mechanismwhereby IFNαβ, following combined TLR (3, 7, or 9)/CD40-agonistimmunization, influences the CD8⁺ T cell response by performing one ormore of the following functions: i) directly augmenting the CD8⁺ T cellresponse to CD70-bearing APCs (CD8 T cell centric), ii) directlyactivating DCs for TNF ligand expression (DC centric), iii) inhibitingregulatory CD4⁺ T cell activity against either APC TNF ligand expressionor of CD8⁺ T cell expansion (Treg centric). Synergistic activity withanti-CD40 in the induction of CD8⁺ T cell expansion is a property of allTLR agonists examined which now includes agonists for TLRs 1/2, 2/6, 3,4, 5, 7, and 9. Collectively, these data demonstrate that combinedTLR/CD40-agonist immunization can reconstitute all of the signalsrequired to elicit potent primary CD8⁺ T cell responses.

To determine the cellular and molecular requirements of the synergybetween the TLRs and CD40, numerous experiments were performed inknockout and/or mice depleted of various cell types or factors byblocking or depletion with antibodies. These studies confirmed thenecessity of intact CD40 and TLR signaling pathways (using CD40 KO andMyD88 KO mice). Though this synergy was not dependent on CD4 cells,IFNγ, IL-12, or IL-23, observed was a variable dependence of the synergyon IFNαβ depending on the TLR agonist used. Ahonen, C. L., C. L. Doxsee,S. M. McGurran, T. R. Riter, W. F. Wade, R. J. Barth, J. P. Vasilakos,R. J. Noelle, and R. M. Kedl. 2004. Combined TLR and CD40 triggeringinduces potent CD8+ T cell expansion with variable dependence on type IIFN. J Exp Med 199.775. It was observed that the degree of dependence onIFNα generally seemed to correlate with the amount of IFNαβ the givenTLR induced. Thus, IFNαβ receptor knockout (IFNαβR KO) mice immunizedwith anti-CD40 in combination with an agonist for TLR 3, 7, or 9 failedto generate a CD8+ T cell response. Conversely, IFNαβR KO mice immunizedwith anti-CD40 in combination with an agonist for TLR 2 or 5 didgenerate a CD8⁺ T cell response. These data suggested to the inventorsthat IFNαβ potentially can play a much larger role in generatingadaptive immunity than has been previously appreciated as shown in theexamples which follow.

At the outset it should be emphasized that the precise role of IFNαβ inthe generation of T cell responses was difficult to predict and clarify.This difficulty is due in part to the fact that many of the effects ofIFNαβ on T cell function appear to be indirect. IFNαβ enhances numerousaspects of APC activation, including the elevation of MHC molecules onthe majority of cell types. Tough, D. F. 2004. Type I interferon as alink between innate and adaptive immunity through dendritic cellstimulation. Leuk Lymphoma 45:257, Le Bon, A., and D. F. Tough. 2002.Links between innate and adaptive immunity via type I interferon. CurrOpin Immunol 14:432 More recently, IFNαβ has been shown to promote APCprocessing of exogenous antigen into the class I pathway, a processknown as cross-priming. Le Bon, A., N. Etchart, C. Rossmann, M. Ashton,S. Hou, D. Gewert, P. Borrow, and D. F. Tough. 2003. Cross-priming ofCD8+ T cells stimulated by virus-induced type I interferon. Nat Immunol4:1009. This allows the generation of CD8⁺ T cell responses after theadministration of exogenous protein antigen. IFNαβ also has othereffects on T cell activation and proliferation. High levels of IFNαβalso induce partial activation of naïve, and proliferation of memory,CD8 T cells. Tough, D. F., S. Sun, X. Zhang, and J. Sprent. 1999.Stimulation of naïve and memory T cells by cytokines. Immunol Rev 170:39Sprent, J., X. Zhang, S. Sun, and D. Tough. 2000. T-cell proliferationin vivo and the role of cytokines. Philos Trans R Soc Lond B Biol Sci355:317, Sprent, J. 2003. Turnover of memory-phenotype CD8+ T cells.Microbes Infect 5:227, Zhang, X., S. Sun, I. Hwang, D. F. Tough, and J.Sprent. 1998. Potent and selective stimulation of memory-phenotype CD8+T cells in vivo by IL-15. Immunity 8:597, Tough, D. F., and J. Sprent.1998. Bystander stimulation of T cells in vivo by cytokines. Vet ImmunolImmunopathol 63:123

The effects of IFNαβ on naïve T cells may in part be mediated throughAPCs, although IFNαβ directly stimulates naïve T cell survival. Marrack,P., J. Kappler, and T. Mitchell. 1999. Type I interferons keep activatedT cells alive. J Exp Med 189:527, Marrack, P., T. Mitchell, J. Bender,D. Hildeman, R. Kedl, K. Teague, and J. Kappler. 1998. T-cell survival.Immuno Rev 165:279. This survival activity is dependent on STAT1 in theT cells, indicating that direct IFNαβ signaling in the T cells must beinvolved. Marrack, P., J. Kappler, and T. Mitchell. 1999. Type Iinterferons keep activated T cells alive. J Exp Med 189:521. Morerecently, IFNα has been show to act directly on naïve CD8⁺ T cells, inconcert with antigen and B7-mediated co-stimulation, to facilitateproliferation, effector function and development of memory Curtsinger,J. M., J. O. Valenzuela, P. Agarwal, D. Lins, and M. F. Mescher. 2005.

Type I IFNs provide a third signal to CD8 T cells to stimulate clonalexpansion and differentiation. J Immunol 174:4465. By contrast, othershave demonstrated that the influence of IFNαβ on the proliferation ofCD8⁺ memory T cells is indirect. This proliferation occurs viaproduction of IL-15 from other cell types, and selectively inducesproliferation of memory CD8 but not CD4 T cells. Zhang, X., S. Sun, I.Hwang, D. F. Tough, and J. Sprent. 1998. Potent and selectivestimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity8:597, Sprent, J., X. Zhang, S. Sun, and D. Tough. 1999. T-cell turnoverin vivo and the role of cytokines. Immunol Lett 65:21. Therefore in theinitiation of T cell activation and proliferation, both indirect anddirect effects of IFNαβ on T cells have been observed.

By contrast, there is little data on the influence of type I IFN onregulatory T cell development or function. One report demonstrated thathuman regulatory cells could be produced in vitro using a combination ofIFNα and IL-10. Levings, M. K., R. Sangregorio, F. Galbiati, S.Squadrone, R. de Waal Malefyt, and M. G. Roncarolo. 2001. IFN-alpha andIL-10 induce the differentiation of human type 1 T regulatory cells. JImmunol 166:5530.

As described above and supported by the data in the examples whichfollow the inventive discovery that type 1 interferon and CD40 agonistcombinations elicit a synergistic effect on cellular immunity andupregulate CD70 on dendritic cells and provide for exponential expansionof CD8+ T cells allows for the development of more potent vaccinesagainst the kinds of diseases whose treatment seems to require thequantity and quality of cellular immunity that the subject noveladjuvant combinations elicit.

The following examples are offered for purposes of exemplification. Itshould be understood, however, that the scope of the present inventionis defined by the claims.

MATERIALS AND METHOD USED IN SOME OF THE FOLLOWING EXAMPLES

C57BL/6, IFNαβR KO, or CD4-depleted IFNαβR KO mice are immunized with amodel antigen. Briefly, 0.1-0.5 mgs of whole protein (ovalbumin or HSVglycoprotein B [HSVgB]) or 50 ug of peptide (SIINFEKL for ovalbumin,SSIFFARL for HSVgB, TSYKSEFV for vaccinia virus B8R) is injected i.p. incombination with a TLR agonist (50 ug Pam3Cys, 25 μg MALP-2, 100 μgPolylC, 150 μg 27609, 50 μg CpG 1826, or 25 μg flagellin), the anti-CD40antibody FGK45 (50 μg), or both. Ovalbumin is purchased from SigmaCorporation (St. Louis, Mo.) and contaminating LPS removed using aTritonX-114 LPS-detoxification methodology as previously described.Adam, O., A. Vercellone, F. Paul, P. F. Monsan, and G. Puzo. 1995. Anondegradative route for the removal of endotoxin fromexopolysaccharides. Anal Biochem 225:321. Whole HSVgB protein is made byexpression in baculovirus and purification on a nickel column, aspreviously described and kindly provided by Dr. Roselyn Eisenberg fromthe University of Pennsylvania. Bender, F. C., J. C. Whitbeck, M. Poncede Leon, H. Lou, R. J. Eisenberg, and G. H. Cohen. 2003. Specificassociation of glycoprotein B with lipid rafts during herpes simplexvirus entry. J Virol 77:9542. The TLR agonists used are either purchased(Pam3Cys-InVivogen, MALP-2-Alexis Biochemicals, PolylC-Amersham/GEHealthcare, CpG 1826-Invitrogen), provided through a material transferagreement (27609-3M Pharmaceuticals), or synthesized in house(flagellin). Each TLR agonist has been tested for LPS contamination byLimulus assay and found to have less than 5 IU of LPS activity(approximately 50-300 ng) for the amounts injected in vivo Injection ofthis amount of LPS has no observable effects on spleen dendritic cellsin vivo (data not shown). In the case of the flagellin isolatedin-house, contaminating LPS was removed using the same protocol asdescribed above for ovalbumin detoxification.

These TLR agonists were chosen for use in our experiments for two mainreasons. First, the major DC subsets in secondary lymphoid tissue arethe CD8⁺ and CD11b⁺ DCs and they express both common and unique TLRs.The TLR agonists chosen directly stimulate either the CD8⁺ DC(polylC-TLR3), the CD11b⁺ DC (27609-TLR7 and flagellin-TLR5), or both DCsubsets (Pam3Cys/MALP-2, TLR2 stimulation). Second, the moleculesselected represent TLR agonists that are either IFNαβ-dependent (polyIC, 27609, CpG 1826) or -independent (Malp-2, Pam3Cys, flagellin) forinducing CD8⁺ T cell responses in combination with anti-CD40.

The immunizations described are performed both with and without theco-administration of the antibodies blocking CD70 (FR70), OX40L/CD134(RM134L), or 41BBL/CD137L (TKS-1). I.p. administration of 250 ug ofantibody every 2 days is sufficient to block the interaction of each ofthese ligand/receptor interactions (See FIG. 5). Blocking experimentsare performed using this regimen and later similar experiments used todetermine the minimum amount of blocking antibody necessary to have aneffect, if any, on the CD8⁺ T cell response.

To monitor the antigen-specific CD8⁺ T cell response, 5-7 days afterimmunization peripheral blood and/or spleen cells are isolated andstained with H-2 Kb/SIINFEKL or H-2K^(b)/SSIFFARL MHC tetramers, aspreviously described. Kedl, R. M., M. Jordan, T. Potter, J. Kappler, P.Marrack, and S. Dow. 2001. CD40 stimulation accelerates deletion oftumor-specific CD8(+) T cells in the absence of tumor-antigenvaccination. Proc Natl Acad Sci USA 98:10811, Kedl, R. M., W. A. Rees,D. A. Hildeman, B. Schaefer, T. Mitchell, J. Kappler, and P. Marrack.2000. T Cells Compete for Access to Antigen-bearing Antigen-presentingCells. J. Ep. Med. 192:1105, Kedl, R. M., B. C. Schaefer, J. W. Kappler,and P. Marrack. 2002. T cells down-modulate peptide-MHC complexes onAPCs in vivo. 3:72. The CD8⁺ T cells are analyzed by intracellularinterferon γ (IC IFNγ) staining as an indicator of the cells' effectorcytokine production capability. IC IFNγ staining has been extensivelyutilized in the literature and will be performed as described. Inaddition, CD107a expression after antigenic stimulation will be analyzedas an indication of antigen-specific lytic function. CD107a (LAMP-1) isa membrane protein constituent of lytic granules and its identificationon the plasma membrane of T cells after antigenic stimulation is anindication of the exocytosis of lytic granules. Combined tetramer andCD107a staining is performed as previously described. Briefly, cells areincubated for 30 minutes with MHC tetramer at 37 degrees. Antigenicpeptide (1 ug/ml) and anti-CD107a-FITC antibody are then added foranother hour, after which 1 ug/ml monensin is added to the cells toinhibit the destruction of the FITC fluorescence as antibody boundCD107a is internalized into lysosomes. The cells are further incubatedfor another 3-4 hours at 37 degrees, stained with antibodies againstCD8, washed, fixed and analyzed by FACS. As described above, IFNαβR KOmice are similarly injected with blocking antibodies to CD70, 41BBL,OX-40L, and CD30L during combined TLR2-or-5/CD40-agonist immunization.The magnitude and function of the CD8+ T cell response will bedetermined by tetramer and IC IFNγ staining and FACS analysis of PBLsand/or spleen cells as described above.

In order to determine the effects of TNF ligand blockade during theprimary immunization on the development of memory CD8⁺ T cells,immunized mice are rested for at least 60 days, re-challenged with thesame immunization, and the secondary response analyzed as describedabove. Experiments are performed in IFNαβR KO mice, CD4-depleted IFNαβRKO mice, and normal and CD4-depleted B6 mice as controls. The TLR/CD40combinations that generate IFNαβ-independent CD8+ T cell responses areanalyzed in the intact IFNαβR KO mice. Both IFNαβ-dependent and-independent TLR/CD40 combinations are tested in CD4-depleted IFNαβR KOmice. Representative CD4-depleted and immunized mice are rested for atleast 60 days after primary immunization and then rechallenged bycombined TLR/CD40-agonist immunization. These experiments are used todetermine whether the primary and memory CD8⁺ T cell response followingimmunization of a IFNαβ-deficient host, CD4-depleted or not, isdependent on CD70 and/or other TNF ligands.

Example 1 CD8+ T Cell Expansion Following Combined TLR(CD40-AgonistImmunization Demonstrates Variable Dependence Upon IFNαβ

While all TLR agonists synergized with anti-CD40 to promote CD8⁺ T cellexpansion, the inventors observed that the CD8⁺ T cell responseselicited from certain TLR agonists/anti-CD40 combinations was completelydependent on IFNαβ. Based thereon the inventors immunized interferon areceptor knockout (IFNαβR KO) mice with peptide antigen in the contextof different combined TLR/CD40-agonists in the experiments contained inFIGS. 1 and 2 as described above.

In the experiment contained in FIG. 1 CD8+ T cell expansion was measuredfollowing combined TLR/CD40 agonist administration in agonmice (bottomrow) which were immunized with ovalbumin peptide, anti-CD40, and theindicated TLR agonists. Seven days later the ovalbumin-specific T cellresponses were measured in the spleen by tetramer staining and FACSanalysis. Numbers in the upper right quadrant indicate the percentage oftetramer staining cells out of total CD8+ cells.

In the experiment contained in FIG. 2 it was shown that CD4 depletion ofIFN alphabetaR KO hosts restores the CD8+ T cell response followingimmunization with IFNalphabeta-dependent TLR agonist in combination withagonistic anti-CD40. WT and IFNalphabetaR KO mice, CD4 depleted ornon-deleted as shown in FIG. 2, were immunized with HSV-1 peptide,agonistic anti-CD40-antibody, and poly IC. Seven days later the HSV-1specific response was determined by tetramer (A) and IC IFNgamma (B)staining PBL cells.

As shown by the results contained in FIG. 2, the CD8⁺ T cell response toimmunization with TLR 3, 7, or δ agonists in combination with anti-CD40was completely abrogated in these mice (FIG. 2). By contrast, the CD8⁺ Tcell response to the remaining TLR/CD40-agonist combinations was eitheronly partially dependent (TLR4/CD40) or relative independent(TLR2/6/CD40-agonists) of IFNαβ (FIG. 1). In other experiments, theTLR1/2 agonist Pam3Cys and the TLR5 agonist flagellin also generatedCD8⁺ T cell responses in IFNαβR KO comparable to wt mice when used incombination with anti-CD40 (data not shown). These results demonstratethat anti-CD40 in combination with a TLR 2 or 5 agonist elicitsIFNαβ-independent CD8⁺ T cell responses while anti-CD40 in combinationwith a TLR 3, 7, or 9 agonist elicits IFNαβ-dependent CD8⁺ T cellresponses. Thus, TLR 2 or 5-agonist synergy with the CD40 pathway can beconsidered IFNαβ-independent. Conversely, TLR 3, 7, or 9-agonist synergywith the CD40 pathway can be considered IFNαβ-dependent. This datasuggested to the inventors a role for IFNαβ in the generation of CD8⁺ Tcell responses by signaling through either the T cells directly, theantigen bearing APC, or both.

Example 2 CD8+ T Cell Expansion Following Combined TLR/CD40-AgonistImmunization is Recovered in CD4-Depleted IFNαβR KO Hosts

The deficient CD8⁺ T cell response in IFNαβR KO mice seemed to suggestto the inventors an obligate role for IFNαβ in the response elicited bycertain TLR/CD40-agonist combinations described above. As shown in theexperiment in FIG. 2, Wt and IFNαβR KO mice were depleted of CD4⁺ Tcells by injection of the anti-CD4 antibody GK1.5 one day before peptideimmunization in conjunction with combined TLR/CD40-agonists (FIG. 2).Seven days after combined TLR/CD40-agonist immunization, mice weresacrificed and PBL and spleen cells were isolated and analyzed bytetramer and intracellular IFNγ staining. Immunization with peptide andpolylC/anti-CD40 failed to generate a CD8⁺ T cell response in IFNαβR KOmice. However, CD4 depletion restored the CD8⁺ T cell response in IFNαβRKO mice with respect to both numbers (percent of total CD8+ T cells,FIG. 2A) and function (FIG. 2B) of antigen-specific T cells. This wastrue for all TLR/CD40-agonist combinations tested (TLRs, 2, 5 and 7)where even the CD8⁺ T cell response to IFNαβ-independentTLR/CD40-agonist combinations (i.e. TLR2) was enhanced compared tonon-CD4-depleted controls (data not shown). Thus, the CD8⁺ T cellresponse in IFNαβR KO mice following combined TLR/CD40-agonistimmunization is generally enhanced after CD4 depletion.

One concern the inventors had with these findings was whether or notthey were physiologically relevant or were simply unique to the IFNαβRKO hosts. Therefore experiments were effected in wt hosts using apolyclonal rabbit anti-IFN antibody to block IFNαβ, with and without CD4depletion.

As shown in FIG. 3 anti-IFN blocks polylC/CD40 mediated CD8 responseswhich are recovered by CD4 depletion. In this experiment mice wereimmunized against ovalbumin (combined polylC/anti-CD40) with and withoutanti-IFN and/or CD4 depletion. at day 7 PBLs were analyzed by tetramerstaining as described in the Materials and Methods above for the percentantigen-specific T cells.

As shown in FIG. 3, for wt mice immunized with combined polylC/αCD40,the anti-FNαβ antibody significantly reduced the magnitude of the CD8+ Tcell response (FIG. 3). Consistent with the results seen in the IFNαβRKO mice, CD4 depletion of anti-IFN treated mice fully recovered the CD8+T cell responses. Therefore, both in the IFNαβR KO hosts as well as inwt host injected with IFNαβ-depleting antibodies, CD4 depletion appearedto alleviate the dependency of the CD8+ T cell response on IFNαβfollowing combined TLR/CD40-agonist immunization. These resultssuggested to the inventors 1) that a subpopulation of CD4⁺ T cellsregulates the CD8⁺ T cell response in IFNαβR KO mice followingimmunization with certain TLR/CD40-agonist combinations, 2) that IFNαβmay play a role in inhibiting the regulatory ability of this populationof CD4⁺ T cells following immunization with these TLR/CD40-agonistcombinations, and 3) other TLR/CD40-agonist combinations (e.g. TLR2 or5) are able to avoid inhibition by regulatory CD4⁺ T cells in aIFNαβ-independent fashion.

These results demonstrate that combined TLR/CD40-agonist immunization isable to elicit potent primary and secondary CD8⁺ T cell responses thatdisplay an intriguing variable dependence on IFNαβ depending upon theTLR agonist utilized. These findings suggested to the inventors a moredirect role for IFNαβ in CD8⁺ T cell responses than has been previouslyappreciated. It was also shown that combined TLR/CD40 agonistimmunization uniquely induces the upregulation of CD70 on DCs, uponwhich the ensuing CD8⁺ T cell response in WT mice appears to be largelydependent. This preliminary data suggested that the increased expressionof CD70 on activated APCs, and the subsequent stimulation ofantigen-specific T cells through CD27, is a primary checkpoint for theformation and survival of CD8⁺ T cell responses in response to combinedTLR/CD40-agonist immunization. More surprising however is ourobservation that IFNαβ-dependent CD8+ T cell responses, in both IFNαβRKO (FIG. 2) and WT mice (FIG. 3), can be rescued by depleting the hostof CD4+ T cells. These results suggested that IFNαβ may influence theCD8⁺ T cell response for the following reasons: 1) regulating the CD8+ Tcell response to TNFL expressing APCs, 2) regulating APC activation andTNF ligand expression, 3) inhibiting CD4⁺ T cell regulatory functionthat suppresses either APC expression of TNF ligands or CD8+ T cellexpansion to the TNFL-bearing APCs, 4) a combination of any of theabove. The examples which follow conclusively determine the accuracy ofthese hypotheses by systematically examining i) the role of IFNαβ inmediating the CD8⁺ T cell response, ii) the role of IFNαβ in DCactivation, and iii) the role of IFNαβ in CD4⁺ regulatory cell function,all following combined TLR/CD40-agonist immunization.

Example 3 Role of TNF Ligands for the CD8+ Response in IFNαβR KOs

As shown in the experiment contained in FIG. 4, the CD8+ T cell responsein WT mice generated by combined TLR/CD40-agonist immunization isdependent on CD70 (See FIG. 4). In this experiment the CD8+ T cellresponse was assayed in CD4-depleted IFNalphabetaR KO hosts followingcombined TLR/CD40 immunization and was shown from the results to belargely dependent on CD70. IFNalphabetaR KO mice were depleted of CD4cells and immunized with HSV-1 peptide, polylC, and anti-CD40 antibodyas described above. Mice were then injected with anti-TNF ligandantibodies as in FIG. 1. At day seven PBLs were again analyzed bytetramer staining.

As shown in the foregoing experiments, the CD8⁺ T cell response inIFNαβR KO mice is unique in that it can only be elicited byTLR/CD40-agonist combinations that do not stimulate IFNαβ, or byCD4-depleting the IFNαβRKO host prior to TLR/CD40-agonist immunization.The results in FIG. 4 further indicate that CD70 plays a necessary rolein the CD8⁺ T cell response in IFNαβR KO mice. It is noted that whileanti-CD70 blocked the response by approximately 10-fold in thisexperiment, other TNFL antibodies inhibited the CD8⁺ T cell responseonly up to 2-fold. This suggests that, in contrast to wt mice (FIG. 1)multiple TNF ligands may have at least some influence on the magnitudeof the CD8⁺ T cell response in IFNαβR KO mice. The data shown in FIG. 4was achieved with minimal blocking antibody injected.

Example 4 Materials and Methods

Injection of a soluble CD70/Ig fusion protein (sCD70Ig), originallydescribed by Dr. Aymen Al-Shamkhani at Southampton General Hospital.(Rowley, T. F., and A. Al-Shamkhani. 2004. Stimulation by soluble CD70promotes strong primary and secondary CD8+ cytotoxic T cell responses invivo. J Immunol 172:6039), successfully provides an agonistic stimulusto T cells through CD27 in vivo. This reagent, kindly provided by Dr.Al-Shamkhani will be injected into IFNαβR KO hosts in combination withTLR and CD40 stimulation. Initially, we will attempt to rescue the CD8+T cell response to IFNαβ-dependent TLR/CD40-agonist combinations by theadditional injection of the sCD70Ig reagent. The CD8+ T cell responsewill again be analyzed on day 7 after initial antigen challenge. Datafrom Dr. Al-Shamkhani's laboratory have determined that daily injectionof 250 ug sCD70Ig on days 2-4 after antigen challenge provide optimalCD70 mediated signals for CD8⁺ T cell expansion (personalcommunication). We have confirmed that this time course of sCD70Iginjection augments the CD8⁺ T cell response to a TLR agonist alone in WTmice (data not shown). Mice will be challenged i.p. on day 0 withantigen and a TLR agonist, anti-CD40, or both. On days two, three, andfour after antigen injection, we will inject 250 ug of sCD70Ig i.p. andthen analyze the CD8⁺ T cell response in the blood and/or spleen 7 daysafter the original antigen challenge.

From the data shown in FIG. 4, it is clear that CD4 depletion of IFNαβRKO hosts makes them responsive to any combination of TLR/CD40stimulation. As shown in FIG. 4 CD70 blockade eliminates the synergybetween the TLR agonist and the CD40 agonist for inducing a CD8+ T cellresponse. In the experiment mice were challenged with the indicatedcombinations of anti-CD40+/TLR− agonist. Representative subsets of micewere injected with the anti-CD70 blocking antibody FR70 (lower dotplots). FIG. 4A shows representative tetramer staining and FIG. 4B showsaverage and standard deviation of 3 mice per group and FIG. 4C showswhere mice were immunized as in 5A but were given none, 1 or 2injections of anti-CD70. DCs were isolated at 24 hours and analyzed forDC numbers (top panels) and CD70 staining (bottom panels) in eachsubset.

It can be seen that the CD8+ T cell response, in WT mice, followingcombined TLR/CD40-agonist immunization is dependent on CD70 (FIG. 4).The data described above and shown in FIG. 4 suggest that this is alsotrue for at least CD4-depleted IFNαβR KO hosts. The results in FIG. 4also suggest that multiple TNF ligands may participate, to one degree oranother, in the CD8+ T cell response in IFNαβR KO hosts.

Example 5 Immune Cell Response Following Recombinant IFNα+/−Anti-CD40 inwt Mice

Experiments were effected using the following materials and methods inorder to determine whether the action of IFNαβ is alone sufficient foreliciting CD8⁺ T cell expansion following immunization withIFNαβ-dependent TLR/CD40-agonist combinations.

Materials and Methods.

Briefly, a novel IFNα sequence was cloned from polylC-stimulated B cellcDNA. Of the induced subtypes, this IFNα subtype was selected because ithas no glycosylation sequences and can therefore be expressed in insectcells without concern for aberrant glycosylation. A TCR Cα epitope tagwas added to the C-terminus for affinity purification purposes and thesequence was cloned into the p10 promoter site of the pBac vector(Invitrogen). Recombinant baculovirus was produced and after infectionof Hi5 cells, recombinant IFNα was purified from the supernatant byaffinity and size chromatography. The activity of the IFNα was confirmedin vivo and in vivo based on the upregulation of class I MHC on APCs(data not shown).

The use of recombinant IFNα in a vaccine setting has been previouslypublished (Le Bon, A., and D. F. Tough. 2002. Links between innate andadaptive immunity via type I interferon. Curr Opin Immunol 14:432) and asimilar protocol will initially be used in the studies proposed here.Wild type mice are primed with antigen and anti-CD40 as described abovein conjunction with 10⁴-10⁶ units of IFNα. The resulting CD8⁺ T cellresponse is then compared to mice immunize with combined TLR(3, 7, or9)/CD40-agonists to determine if IFNα can synergize with anti-CD40 tothe same degree as TLR stimulation for eliciting CD8⁺ T cell expansion.Other control mice are injected with IFNα or anti-CD40 only. CD8+ T cellresponses are analyzed as described above.

As shown in the experiment contained in FIG. 5, the data obtainedrevealed that there is a synergistic effect on immunity with recombinantIFNα and anti-CD40. Mice were immunized with antigen in the context of 3injections of 1×10⁵ units IFN, a single injection of 1×10⁶ units IFN,anti-CD40 alone, or anti-CD40 in conjunction with either dosing regimenof IFN. While IFN or CD40 alone stimulated a detectable CD8+ T cellresponse, the combined IFN/CD40 synergized to produce a CD8+ T cellresponse similar to that observed in response to polylC/CD40immunization (FIG. 5).

More particularly, this experiment reveals that the combinedadministration of type 1 interferon and an agonistic CD40 antibodyinduced an exponential expansion of antigen specific CD8+ and T cellscompared to administration of either alone. Mice were injected i.p. withovalbumin and the indicated combinations of anti-CD40, poly IC, orrecombinant IFN. For IFN injections, mice were either given 3consecutive daily injections of 1×10⁵ units IFN, starting on the day ofantigen injection, or a single injection of 1×10⁶ units IFN at the sametime of antigen injection. Seven days later, the mice were sacrificedand cells from either peripheral blood or spleen were stained withTetramer to identify the magnitude of expansion of ovalbumin specificCD8+ T cells. The cells were analyzed by FACS and the data shown wasgated on CD8+ B220-events. In the FIG. 5-(A) is the dot plates oftetramer staining and 5(B) is the average and standard deviation (from 2individual mice) of two percent tetramer and CD8+ cell in the blood outof total CD8+ T cells.

The data contained in FIG. 5 reveals that recombinant type 1 interferonsynergizes with CD40 to a similar degree as TLR/CD40 stimulation theseresults further demonstrate that the recombinant IFN produced inbaculovirus works well in vivo. Moreover, these results reveal thatcombined IFNα/CD40 stimulation can synergize to a similar magnitude asTLR/CD40 stimulation in promoting CD8+ T cell expansion.

Example 6 Combined Administration of Type 1 Interferon and CD40 AntibodyInduce CD70+Expression on DCs

The data contained in the afore-described experiments suggests thatIFNαβ-dependency is determined by the response of the DC and/or CD4+Tregs to IFNαβ. The inventors hypothesized that CD70 is involved in themechanism by which IFNαβ, in the context of combinedIFNalpha/CD40-agonist immunization, elicits such potent CD8⁺ T cellimmunity. The results of the prior example particularly reveal that CD40agonist and type 1 interferon elicit a synergistic effect on CD8+immunity. (See FIG. 5). This data shows the eventual effect of combinedIFNα/CD40 stimulation on the responding CD8+ T cells, not the APCs. Thefollowing experiments are conducted to examine whether combinedIFNα/CD40 stimulation induces the expression of CD70 and/or other TNFligands on antigen-bearing DC subsets.

Using the recombinant IFNα described above iWT B6 mice are primed withantigen and anti-CD40 as described above in conjunction with 10⁴-10⁶units of IFNα. As controls, mice are immunized with anti-CD40 alone,IFNα alone, or combined polylC/anti-CD40 positive control for theincrease in DC CD70 expression. Representative mice are sacrificed 6-48hours after priming, the spleens collagenase digested, and the DCsstained and analyzed by FACS. The DCs are assessed for their expressionof the TNF ligands CD70, 41 BBL, OX-40L, CD30L, and GITRL. The resultingDC phenotype is compared to mice immunized with combined TLR3, 7, or9/CD40-agonists to determine if IFNα can synergize with anti-CD40 to thesame degree as TLR stimulation for eliciting CD8+ T cell expansion.Other control mice will be injected with IFNα or anti-CD40 only. Todetermine the influence of IFNα on antigen processing and presentationof the various subsets, mice are challenged with fluorescent antigen asdescribed above in conjunction with recombinant IFNα+/−anti-CD40.Antigen uptake, antigen presentation, and DC activation and TNFLexpression are determined as described above. These experimentsdetermine how IFNα, independently and in conjunction with anti-CD40,influences antigen presentation, DC TNFL expression, and CD8⁺ T cellexpansion.

As shown in the experiment contained in FIG. 6 the combinedadministration of type 1 interferon and an agonistic CD40 antibodyinduces CD70 expression on CD8+ T cells in vivo whereas theadministration of either alone does not. In the experiment mice wereinjected with anti-CD40 antibody alone, polylC (positive control),recombinant alpha interferon or anti-CD40 antibody and type 1interferon. Eighteen hours later spleen DCs were isolated and analyzedfor their expression of CD70. The numbers in the upper right quadrant ofFIG. 7 indicate the mean fluorescent intensity of CD70 staining. Thisdata also reveals that similar to polylC/CD40 agonist administration,CD40/IFN similarly increases the expression of CD70 on CD8+ DCs.

Therefore, the data (FIG. 6) demonstrates the success of IFNα/CD40immunization at eliciting a CD8+ T cell response and show that the DCsin IFNα/anti-CD40 injected mice are similar to DCs from combinedTLR/CD40-agonist immunized controls with respect to antigen uptake,antigen presentation, and/or TNFL upregulation. Specifically, CD70 isincreased on one or more DC subsets following combined IFNα/αCD40immunization, though not with challenge of either stimulus alone.

Example 7 Combined Administration of Increasing Amounts of Alpha IFNwith and without CD40 Agonistic Antibody

In the experiment contained in FIG. 7, mice were injected as in theforegoing example, but with increasing amounts of type 1 interferon andin the presence and absence of the agonistic CD40 antibody. The data inthe Figure is expressed as the average CD70 MFI between two individualmice and the error bars represent standard deviation. These resultssimilarly reveal that the combined administration of the type 1interferon and the CD40 agonist increased CD70 expression on DCs in vivowhereas the type 1 interferon and the CD40 agonist, when each wereadministered in the absence of the other did not.

Example 8 Percentage of Antigen Specific T Cells in Mice Immunized withDecreasing Doses of IFN Alpha and CD40 Agonist or Anti-CD70

In this experiment contained in FIG. 8, mice were immunized withanti-CD40 antibody, IFN alpha and anti-CD40 antibody at differentdecreasing doses as set forth therein, polylC and CD40 antibody, andalpha interferon and anti-CD40 antibody. It can be seen from the datacontained therein that the number of antigen (ovalbumin) specific Tcells decreased exponentially with the lower IFN alpha dosages and thatnumber of antigen specific cells with the IFN/polylC and IFNalpha/CD40agonist were substantially the same. (See FIG. 8) Thus, the data inFIGS. 6 and 7 and 8 shows that exogenously added IFNα can synergize withanti-CD40 and upregulate CD70 expression on DCs and result in theexpansion of antigen specific T cells.

Example 9 CD70 Expression on DCs from IFNalphabetaR KO Mice withTLR/CD40 Agonist Combination

In order to substantiate that the results seen in FIGS. 6 and 7 withexogenously added IFN alpha correlate to endogenous IFN, experimentswere conducted in IFNαβ R mice as depicted in FIG. 9. As shown therein,experiments are performed wherein mice were successfully reconstitutedwith the transferred bone marrow (in this case, BM expressingGFP+/−Bcl-2, (FIG. 9) and which generated an immune response followingimmunization 8 weeks after reconstitution (not shown).

As shown in the experiment in FIG. 9, combined TLR/CD40 agonistadministration challenge induces CD70 expression only on DCs expressingthe targeted TLR in IFNalphabetaR KO mice. In the experiment,IFNalphabetaR mice were injected with anti-CD40 antibody alone, or incombination with polylC or Pam3Cys. Pam3Cys is a TLR2 agonist and polylCis a TLR3 agonist. 24 hours later, the spleen DCs were isolated andstained for CD70 expression as afore-described. CD8+ DCs express TLR2and TLR3, whereas CD11b+ DCs express TLR2 but not TLR3. These datasuggest that in the absence of IFNalphabeta signaling only DCsstimulated directly through both TLR and CD40 are capable of increasedCD70 expression.

This data in combination with the prior data further suggest that thisincrease in CD70 expression is involved in the concomitant expansion ofCD8+ T cells.

Example 10 Effect of Type 1 IFN/CD40 Combination Versus Effect ofIL-2/CD40 Agonist Combination on Antigen Specific T Cell Numbers

This experiment in FIG. 10 was designed to compare the effect of IL-2another cytokine and a type 1 interferon when combined with a CD40agonist. As noted above, the synergy achieved with the IFNalpha/CD40agonist combination is believed to be truly unexpected and is not seenwith other cytokine/CD40 agonist combinations.

In this experiment the effect of type 1 IFN/CD40 antibody, IL-2/CD40antibody, IL-2 alone, IFNalpha alone, and CD40 agonist alone werecompared. This results contained in FIG. 10 reveal that IL-2 andIFN/CD40 combinations do not yield similar effects on the percentages ofantigen-specific T cell immune cells. Therein, mice were injected withovalbumin (300 mg) in combination with anti-CD40 (50 mg) recombinant IFNalpha (1×10⁶ U), IL-2 (1×10⁶ U) IL-2 and CD40, or the same dosages ofIFN and CD40 agonist alone. Seven days later peripheral blood was takenand stained with Kb/ova tetramer to identify the percentage of antigenspecific T cells. Numbers in the dot plots are the percent of total CD8+T cells in the indicated oval gate (tetramer +). The bar graph is theaverage and standard deviation of 2 mice per injection. The resultstherein show that the number of antigen specific T cells was much higherin the animals administered the IFN/CD40 combination versus theIL-2/CD40 combination, with the same amount of CD40 agonist and whenboth cytokines were administered at the same activity levels. Thisfurther substantiates that the synergy achieved with the IFN/CD40agonist combination is unexpected.

Example 11 Effect of IFNalpha and CD40 Agonist on Survival Time inMetastatic Melanoma

In this experiment C57B116 mice were intravenously inoculated with100,000 B16.F10 melanoma cells on day zero. Four days later, micereceived 100 micrograms tumor pep (deltaV) 100 micrograms of anti-CD40and 1×106 units of alpha interferon. As shown therein the mice whichwere administered the and CD40/IFN combination had a substantiallygreater survival time. This data further supports the potentialapplication of the subject adjuvant combination in tumor vaccines andcancer therapy.

Example 12 Effect of CD40 Agonist/IFNalpha Combination on MetastaticLung Cancer t

The experiment in FIG. 12 shows that the subject CD40 agonist/IFN alphacombination protects mice from metastatic lung cancer. In thisexperiment C57BI/6 mice were intravenously inoculated with 100,000B16.F10 melanoma cells on day zero. Four days later, mice received 100micrograms of tumor peptide, deltaV, 100 micrograms anti-CD40 antibody,100 micrograms of S-27609 (TLR7 agonist) and 1×106 units of alphainterferon. Twenty-one days later post tumor challenge mice weresacrificed, lungs were removed therefrom, and metastatic nodules werecounted via a dissection microscope. In Panel A of the Figure is showndigital pictures of representative lungs at day of lung harvest. Inpanel B is shown an enumeration of lung metastases wherein N=7-8 miceper group. These results show the protective effect of the CD40agonist/IFN alpha combination versus the other treatments.

Example 13 Tumor Infiltrating Analysis from Tumor Bearing Lungs

Experiments shown in FIG. 13 were conducted wherein TIL (tumorinfiltrating lymphocytes) analysis from tumor bearing lungs waseffected. C57BI/6 mice were intravenously inoculated with 100.000B16.F10 melanoma cells on day zero. Five days later, mice received 100micrograms tumor peptide (deltaV), 100 micrograms anti-CD40 and 1×10⁶units of interferon alpha as shown therein. Twenty days post tumorchallenge mice were sacrificed. The lungs were removed and TILs wereisolated via Percoll gradient centrifugation. Cells were subsequentlysubjected to flow cytometric analysis to investigate the relative andabsolute numbers of infiltrating CD4 (13A and 13D), CD8 (13B and 13E)and FoxP3+ cells (13 C and 13F). In the experiment N=4 mice per group.

Example 14 Effect of Combination Immunotherapy on CD8+ T Cells ThatInfiltrate Lungs in Tumor Bearing Mice

In this experiment contained in FIG. 14, the effect of the subjectcombination immunotherapy on the generation of antigen-specific effectorCD8+ T cells that infiltrate lungs of tumor bearing mice was analyzed.In the experiment therein C57BI/6 mice were intravenously inoculatedwith 100,000 B16.F10 melanoma cells on day zero. Five days later, themice received 100 micrograms of the tumor peptide (deltaV), 100micrograms of anti-CD40, and 1×106 units of alpha interferon asindicated. Twenty days post tumor challenge mice were sacrifice andlunges were removed and the TILs were again isolated via Percollgradient centrifugation. Cells were subsequently stimulated with 1microgram/mL rhIL-2 and brefeldin A for 12-18 hours and then subjectedto intracellular cytokine staining. Cells were first labeled withantibodies to CD8 and CD44, then fixed and rendered permeable beforestaining with IFNg. Positive cells were calculated by subtracting thebackground observed with the irrelevant (SIINFEKL) peptide control andthen plotted as either percent positive (14A) or absolute numbers (14B)of CD8+ CD44+IFNg+T cells. In the experiment N=4 mice per group.

The results in the Figure reveal that the number of antigen specificCD8+ T cells is increased as a result of the subject IFN/CD40 agonistcombination administration. These results further substantiate theefficacy of the subject adjuvant combination in cancer vaccines andother therapies wherein such immune potentiation is desired.

As a final note, in order to further describe the invention, thisapplication contains FIG. 15 which contains the sequence of an exemplaryagonistic antibody which was used in the examples as well as FIGS. 16and 17 which depict schematically methods and materials suitable forproducing DNA constructs and polypeptide conjugates according to theinvention, e.g., using a baculovirus expression system.

It is to be understood that the invention is not limited to theembodiments listed hereinabove and the right is reserved to theillustrated embodiments and all modifications coming within the scope ofthe following claims.

The various references to journals, patents, and other publicationswhich are cited herein comprise the state of the art and areincorporated by reference as though fully set forth.

1. A nucleic acid construct comprising: (i) at least one nucleic acidsequence encoding an agonist of CD40; (ii) optionally a nucleic acidsequence encoding a desired antigen; and (iii) a nucleic acid sequenceencoding a type 1 interferon; wherein said sequences (i), (ii) (ifpresent) and (iii) are operably linked to the same or differenttranscription regulatory sequences and further wherein said sequences(i), (ii) and (iii) are optionally separated by a linker sequence and/oran IRES.
 2. The nucleic acid construct of claim 1 wherein thepolypeptide type 1 interferon is selected from interferon α, β, tao,epsilon, zeta and omega.
 3. The nucleic acid construct of claim 1wherein the type 1 interferon is alpha interferon.
 4. The nucleic acidconstruct of claim 3 wherein type 1 interferon is P interferon.
 5. Thenucleic acid construct of claim 1 wherein the CD40 agonist is ananti-CD40 antibody or an agonistic CD40 antibody fragment.
 6. Thenucleic acid construct of claim 1 wherein the CD40 agonist is a CD40L.7. The nucleic acid construct of claim 6 wherein said CD40L is a human,murine, rat, or a primate CD40L.
 8. The nucleic acid construct of claim7 wherein the CD40L is human CD40L or a soluble human CD40L fragment,soluble CD40L oligomer or variant or conjugate of CD40L that binds humanCD40.
 9. The nucleic acid construct of claim 5 wherein said antibody isa chimeric antibody.
 10. The nucleic acid construct of claim 5 whereinsaid antibody is a humanized antibody.
 11. The nucleic acid construct ofclaim 5 wherein said antibody is a human antibody.
 12. The nucleic acidconstruct of claim 5 wherein said anybody is a single chainimmunoglobulin.
 13. The nucleic acid construct of claim 5 wherein saidantibody comprises human heavy and light chain constant regions.
 14. Thenucleic acid construct of claim 5 wherein said antibody is selected fromthe group consisting of an IgG1, IgG2, IgG3 and an IgG4.
 15. The nucleicacid construct of claim 5 wherein said antibody is encoded by animmunoglobulin light chain encoding nucleic acid sequence and animmunoglobulin heavy chain encoding nucleic acid sequence which areoperably linked to the same promoter.
 16. The nucleic acid construct ofclaim 15 wherein said immunoglobulin light chain and immunoglobulinheavy chain sequences are intervened by an IRES.
 17. The nucleic acidconstruct of claim 1 wherein said antigen sequence (ii) encodes a viral,bacterial, fungal, or parasitic antigen.
 18. The nucleic acid constructof claim 1 wherein said antigen sequence (ii) encodes a human antigen.19. The nucleic acid construct of claim 18 wherein said human antigen isa cancer antigen, autoantigen or other human antigen the expression ofwhich correlates or is involved in a chronic human disease.
 20. Thenucleic acid construct of claim 17 wherein said viral antigen isspecific to a virus selected from the group consisting of HIV, herpes,papillomavirus, ebola, picorna, enterovirus, measles virus, mumps virus,bird flu virus, rabies virus, VSV, dengue virus, hepatitis virus,rhinovirus, yellow fever virus, bunga virus, polyoma virus, coronavirus,rubella virus, echovirus, pox virus, varicella zoster, African swinefever virus, influenza virus and parainfluenza virus.
 21. The nucleicacid construct of claim 3 wherein said a interferon is human ainterferon which optionally may be PEGylated.
 22. The nucleic acidconstruct of claim 17 wherein said bacterial antigen is derived from abacterium selected from the group consisting of Salmonella, Escherichia,Pseudomonas, Bacillus, Vibrio, Campylobacter, Heliobacter, Erwinia,Borrelia, Pelobacter, Clostridium, Serratia, Xanothomonas, Yersinia,Burkholdia, Listeria, Shigella, Pasteurella, Enterobacter,Corynebacterium and Streptococcus.
 23. The nucleic acid construct ofclaim 17 wherein said parasite antigen is derived from a parasiteselected from Babesia, Entomoeba, Leishmania, Plasmodium, Trypanosoma,Toxoplasma, Giarda, flat worms and round worms.
 24. The nucleic acidconstruct of claim 17 wherein said fungal antigen is derived from afungi selected from the group consisting of Aspergillus, Coccidoides,Cryptococcus, Candida Nocardia, Pneumocystis, and Chlamydia.
 25. Thenucleic acid construct of claim 1 wherein the antigen is tumor antigen.26. The nucleic acid construct of claim 25 wherein said tumor antigen isa lung tumor antigen.
 27. The nucleic acid construct of claim 1 whereinthe antigen is a cancer antigen expressed by a human cancer selectedfrom the group consisting of prostate cancer, pancreatic cancer, braincancer, lung cancer (small or large cell), bone cancer, stomach cancer,liver cancer, breast cancer, ovarian cancer, testicular cancer, skincancer, lymphoma, leukemia, colon cancer, thyroid cancer, cervicalcancer, head and neck cancer, sarcoma, glial cancer, and gall bladdercancer
 28. The nucleic acid construct of claim 1 wherein the antigen isan autoantigen the expression of which correlates to an autoimmunedisease.
 29. An expression vector containing a nucleic acid constructaccording to claim
 1. 30. The expression vector of claim 29 which isselected from a plasmid, recombinant virus, and episomal vector
 31. Arecombinant host cell or non-human animal which expresses a nucleic acidconstruct according to claim
 1. 32. The recombinant host cell of claim31 which is selected from bacterial cell, yeast cell, mammalian cells,insect cells, avian cells and amphibian cells.
 33. The recombinant hostcell of claim 31 which is a human cell.
 34. A protein conjugate thatresults upon expression of the nucleic acid construct or vectoraccording to any one of claims 1-30.
 35. The protein conjugate of claim34 which comprises an anti-CD40 antibody, human alpha interferon, and anantigen the expression of which correlates to a disease condition. 36.The protein conjugate of claim 35 wherein said disease is selected fromcancer, allergy, an autoimmune disease, an infectious disease and aninflammatory condition.
 37. The protein conjugate of claim 36 whichcomprises an HIV antigen.
 38. The protein conjugate of claim 37 whereinthe HIV antigen is Gag.
 39. A method for eliciting an enhanced cellularimmune response by administering a nucleic acid construct according toany one of claims 1-28 or a vector or host cell containing said nucleicacid construct.
 40. The method of claim 39 wherein said administeringresults in at least one of the following: (i) enhanced primary andmemory CD8+ T cell responses relative to the administration of a DNAencoding only a CD40 agonist or type 1 interferon; (ii) inducesexponential expansion of antigen specific CD8+ T cells; (iii) generatesa protective immune response in a CD4 deficient host comparable to anormal (non-CD4 deficient) host; and (iv) induction of CD70 expressionon dendritic cells.
 41. The method of claim 39 wherein the enhancedcellular immune response is specific to an antigen selected from a viralantigen, bacterial antigen, fungal antigen, autoantigen, allergen, and acancer antigen.
 42. The method of claim 41 wherein the antigen is a HIVantigen.
 43. The method of claim 42 wherein the HIV antigen is gag orenv.
 44. The method of claim 41 wherein the antigen is an antigenexpressed by a human tumor.
 45. A method for eliciting an enhanced CD8+T cell immune response in a subject in need thereof comprisingadministering synergistically effective amounts of (i) at least one CD40agonist, (ii) at least one type 1 interferon and optionally (iii) atleast one antigen the expression of which is correlated to a specificdisease. or a polypeptide conjugate containing these moieties, whereinthese moieties are comprised in the same or separate pharmaceuticallyacceptable compositions.
 46. The method of claim 45 wherein the CD40agonist, the type 1 interferon and the antigen, if present, arecomprised in the same composition.
 47. The method of claim 45 whereinthe CD40 agonist and the type 1 interferon are comprised in separatecompositions.
 48. The method of claim 45 wherein the CD40 agonist is ananti-CD40 antibody.
 49. The method of claim 45 wherein the CD40 agonistis a CD40L polypeptide.
 50. The method of claim 49 wherein said CD40Lpolypeptide comprises a soluble CD40L polypeptide, fragment thereof oroligomeric CD40L polypeptide or a conjugate containing any of theforegoing.
 51. The method of claim 50 wherein the CD40L comprises humanCD40L or a soluble fragment, oligomer, or conjugate containing thatbinds human CD40.
 52. The method of claim 45 wherein the type 1interferon is selected from alpha, beta, alpha/beta, epsilon, tau, omegaor zeta interferon or a variant or fragment or PEGylated form thereof.53. The method of claim 45 wherein the disease is selected from cancer,allergy, inflammatory disease, infectious disease and an autoimmunedisease.
 54. The method of claim 53 wherein the infectious disease iscaused by a virus, bacterium, fungus, or parasite.
 55. The method ofclaim 54 wherein the virus is HIV.
 56. The method of claim 45 whereinsaid administration results in at least one of the following: (i)elicits substantially enhanced primary and memory CD8+ T cell responsesrelative to the administration of the CD40 agonist or the type 1interferon alone; (ii) induces exponential expansion of antigen specificCD8+ T cells; and (iii) generates a protective immune response in a CD4deficient host that is comparable to a normal (non-CD4 deficient) host;and (iv) induces CD70 expression on dendritic cells.
 57. The method ofclaim 56 which is used to treat a viral infection or cancer.
 58. Themethod of claim 39 wherein the nucleic acid construct is administeredmucosally, topically, orally, intravenously, intramuscularly,intranasally, vaginally, rectally, intratuumorally, intrathecally, orintraocularly.
 59. The method of claim 42 wherein the polypeptideconjugate is administered mucosally, topically, orally, intravenously,intramuscularly, intranasally, vaginally, rectally, intratumorally,intrathecally, or intraocularly.
 60. A composition suitable for use inhuman therapy in eliciting an enhanced CD8+ T cell immune response thatcomprises synergistically effective amounts of (i) at least one CD40agonist, (ii) at least one type 1 interferon and (iii) optionally atleast one antigen.
 61. The composition of claim 60 wherein the CD40agonist is an agonistic anti-CD40 antibody or an agonistic anti-CD40antibody fragment.
 62. The composition of claim 61 wherein the agonisticanti-CD40 antibody is a human, chimeric, humanized, or single chainantibody.
 63. The composition of claim 61 wherein the agonisticanti-CD40 antibody is an IgG1, IgG2, IgG3 or IgG4.
 64. The compositionof claim 60 wherein the CD40 agonist is a CD40L polypeptide.
 65. Thecomposition of claim 64 wherein the CD40L polypeptide is a soluble humanCD40L fragment, oligomer thereof, or conjugate containing.
 66. Thecomposition of claim 60 which comprises a human tumor antigen, orautoantigen.
 67. The composition of claim 60 which comprises abacterial, viral or fungal antigen.
 68. The composition of claim 60wherein the type 1 interferon is a human alpha or beta interferon. 69.The composition of claim 68 wherein said interferon is a consensus alphainterferon or PEGylated alpha or beta interferon.
 70. The composition ofclaim 60 which comprises an allergen.
 71. A method of enhancing theefficacy of a vaccine composition comprising adding or administeringsaid vaccine in conjunction with at least one CD40 agonist and at leastone type 1 interferon.
 72. The method of claim 71 wherein said CD40agonist is a CD40 agonistic antibody or fragment thereof.
 73. The methodof claim 71 wherein the type 1 interferon is a human alpha interferon ora human beta interferon.
 74. The method of claim 71 wherein saidaddition or combined administration induces enhanced CD8+ T cellimmunity specific to an antigen contained in the vaccine.
 75. The methodof claim 74 which induces CD70 expression on dendritic cells.
 76. Amethod for alleviating the toxicity of a CD40 agonist by administeringsaid agonist in conjunction with an amount of a type 1 interferon or aTLR agonist that is sufficient to reduce or eliminate liver toxicityrelative to when the CD40 agonist is administered in the absence of saidtype 1 interferon or TLR agonist.
 77. The method of claim 76 wherein theCD40 agonist is a CD40 agonistic antibody or fragment or a CD40Lpolypeptide.
 78. The method of claim 76 wherein reduced liver toxicityis determined based on liver transaminase levels.
 79. The method ofclaim 76 which provides for a maximum tolerated dose (MTD) for the CD40agonist which is about 1.5-10-fold higher than a MTD which in theabsence of the type 1 interferon or TLR agonist results in the sameincrease in liver transaminase levels.